Methods for treating renal disease

ABSTRACT

The invention features methods for treating or reducing the likelihood of developing a renal disease by administering to a subject in need thereof an agent that decreases expression of a pathogenic APOL1. The agents of the method target various signaling pathways and decrease the level of the pathogenic APOL1 polypeptide.

FIELD OF THE INVENTION

The field of invention is the treatment of renal disease, such as focalsegmental glomerulosclerosis (FSGS), end-stage kidney disease (ESKD) ornon-diabetic chronic kidney disease, in a subject (e.g., a subjecthaving one or more APOL1 risk alleles and expressing a pathogenic APOL1polypeptide) by administering an agent that targets specific signalingpathways and thereby decreases APOL1 polypeptide expression.

BACKGROUND OF THE INVENTION

End-stage kidney failure (ESKD) is a growing problem that now affectsover half a million individuals in the United States. The cost of caringfor patients with ESKD is currently over 40 billion dollars per year. Inthe U.S., the likelihood that subjects of African descent will developESKD is 4 to 5 times higher than for Americans without African ancestry.These facts are reflected in the disparity between the 12-13% of theU.S. population with African descent and the 40% of U.S. dialysispatients who are African-American. The epidemic of renal disease riskfactors, such as obesity and metabolic syndrome, suggests that themagnitude of this problem will only increase.

There are no specific therapies for the vast majority of progressivekidney diseases. Some types of chronic renal disease progression can beslowed by blood pressure control with specific agents, but nephrologistscannot accurately predict which patients will respond. Moreover, whilesuccessful treatment typically slows progression, it neither preventsdisease nor halts disease progression. Recent large trials have shownthat African Americans may derive less benefit from drugs used to slowthe progression of renal disease (Appel et al., N.E. J. Med.363:918-929, 2010). There are few, if any, common diseases that showsuch a marked disparity in the United States.

Recently it was determined that that specific genetic variants thatalter the protein sequence of APOlipoprotein-L1 (APOL1) are present onlyin individuals with recent African ancestry and account for a largeproportion of this major health disparity. Surprisingly, APOL1 kidneydisease variants have a major impact on multiple different types ofkidney disease including hypertension-associated end-stage renal disease(H-ESRD), focal segmental glomerulosclerosis (FSGS), and HIV-associatednephropathy (HIVAN). Individuals with these variant APOL1 alleles have a7-30 fold increased risk for kidney disease. Based on the high frequencyof these APOL1 risk alleles, more than 3.5 million African Americanslikely have the high risk APOL1 genotype. African Americans without thehigh risk genotype have little excess risk compared with Americans ofEuropean ancestry.

Despite evidence that variants in the APOL1 gene cause renal disease,very little is known about the biology of its product, APOL1, or itsrole in the kidney. APOL1 has a defined role in resistance totrypanosomes, and the G1 and G2 variants appear to have become common inAfrica because they confer protection against the forms of trypanosomesthat cause African Sleeping Sickness.

There still exists a need for therapies for kidney diseases in patientswith one or more APOL1 risk alleles, which cause great morbidity andmortality with high economic impact in this and other subjectpopulations.

SUMMARY OF THE INVENTION

The invention features methods for treating renal disease in patientswith a high-risk APOL1 genotype (e.g., those patients having at leastone or more APOL1 risk alleles).

A first aspect of the invention features a method of treating orreducing the likelihood of developing a renal disease by administeringto a subject in need thereof an agent (e.g., an agent that targets theJanus Kinase (JAK)/Signal Transduction Activator of Transcription (STAT)pathway, the toll like receptor (TLR) pathway, the NF-κB pathway, theprotein kinase R (PKR) pathway, the MAP kinase pathway, and the TANKbinding kinase 1 (TBK1)/lκB kinase e (lKKe) pathway) that decreases thelevel of expression of a pathogenic APOL1 encoded by an APOL1 riskallele.

As used herein, the term “alkyl,” “alkenyl” and “alkynyl” includestraight-chain, branched-chain and cyclic monovalent substituents, aswell as combinations of these, containing only C and H whenunsubstituted. Examples include methyl, ethyl, isobutyl, cyclohexyl,cyclopentylethyl, 2-propenyl, 3-butynyl, and the like. The term“cycloalkyl,” as used herein, represents a monovalent saturated orunsaturated non-aromatic cyclic alkyl group having between three to ninecarbons (e.g., a C3-C9 cycloalkyl), unless otherwise specified, and isexemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, bicyclo[2.2.1]heptyl, and the like. When the cycloalkylgroup includes one carbon-carbon double bond, the cycloalkyl group canbe referred to as a “cycloalkenyl” group. Exemplary cycloalkenyl groupsinclude cyclopentenyl, cyclohexenyl, and the like.

Typically, the alkyl, alkenyl and alkynyl groups contain 1-12 carbons(e.g., C1-C12 alkyl) or 2-12 carbons (e.g., C2-C12 alkenyl or C2-C12alkynyl). In some embodiments, the alkyl groups are C1-C8, C1-C6, C1-C4,C1-C3, or C1-C2 alkyl groups; or C2-C8, C2-C6, C2-C4, or C2-C3 alkenylor alkynyl groups. Further, any hydrogen atom on one of these groups canbe replaced with a substituent as described herein.

The term “alkoxy” represents a chemical substituent of formula —OR,where R is an optionally substituted alkyl group (e.g., C1-C6 alkylgroup), unless otherwise specified. In some embodiments, the alkyl groupcan be substituted, e.g., the alkoxy group can have 1, 2, 3, 4, 5, or 6substituent groups as defined herein. Similarly, the term “alkaryloxy”represents a chemical substituent of formula —OR, where R is anoptionally substituted alkaryl group.

“Aromatic” moiety or “aryl” moiety refers to any monocyclic or fusedring bicyclic system which has the characteristics of aromaticity interms of electron distribution throughout the ring system and includes amonocyclic or fused bicyclic moiety such as phenyl or naphthyl;“heteroaromatic” or “heteroaryl” also refers to such monocyclic or fusedbicyclic ring systems containing one or more heteroatoms selected fromO, S, and N. The inclusion of a heteroatom permits inclusion of5-membered rings to be considered aromatic as well as 6-membered rings.Thus, typical aromatic/heteroaromatic systems include pyridyl,pyrimidyl, indolyl, benzimidazolyl, benzotriazolyl, isoquinolyl,quinolyl, benzothiazolyl, benzofuranyl, thienyl, furyl, pyrrolyl,thiazolyl, oxazolyl, isoxazolyl, benzoxazolyl, benzoisoxazolyl,imidazolyl, and the like. Because tautomers are theoretically possible,phthalimido is also considered aromatic. Typically, the ring systemscontain 5-12 ring member atoms or 6-10 ring member atoms.

“Halogen” refers to a halogen atom that is F, Cl, Br, or I, and moreparticularly is fluorine or chlorine.

An “oxo” group is a substituent having the structure C═O, where there isa double bond between a carbon and an oxygen atom. Similarly, a “thioxo”group is a substituent having the structure C═S, where there is a doublebond between a carbon and a sulfur atom.

Typical optional substituents on aromatic or heteroaromatic groupsinclude independently halo (e.g., F, Cl, Br, or I), CN, NO₂, CF₃, OCF₃,COOR′, CONR′₂, OR′, SR′, SOR′, SO₂R′, NR′₂, NR′(CO)R′,NR′C(O)OR′,NR′C(O)NR′₂, NR′SO₂NR′₂, or NR′SO₂R′, wherein each R′ is independently Hor an optionally substituted group selected from alkyl, alkenyl,alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, and aryl(all as defined above); or the substituent may be an optionallysubstituted group selected from alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, aryl, heteroaryl, O-aryl, O-heteroaryl,and arylalkyl.

Optional substituents on a non-aromatic group (e.g., alkyl, alkenyl, andalkynyl groups), are typically selected from the same list ofsubstituents suitable for aromatic or heteroaromatic groups, except asnoted otherwise herein. A non-aromatic group may also include asubstituent selected from ═O and ═NOR′ where R′ is H or an optionallysubstituted group selected from alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteralkynyl, heteroaryl, and aryl (all as definedabove).

In general, a substituent group (e.g., alkyl, alkenyl, alkynyl, or aryl(including all heteroforms defined above) may itself optionally besubstituted by additional substituents. The nature of these substituentsis similar to those recited with regard to the substituents on the basicstructures above. Thus, where an embodiment of a substituent is alkyl,this alkyl may optionally be substituted by the remaining substituentslisted as substituents where this makes chemical sense, and where thisdoes not undermine the size limit of alkyl per se; e.g., alkylsubstituted by alkyl or by alkenyl would simply extend the upper limitof carbon atoms for these embodiments, and is not included. However,alkyl substituted by aryl, amino, halo and the like would be included.For example, where a group is substituted, the group may be substitutedwith 1, 2, 3, 4, 5, or 6 substituents. Optional substituents include,but are not limited to: C1-C6 alkyl or heteroaryl, C2-C6 alkenyl orheteroalkenyl, C2-C6 alkynyl or heteroalkynyl, halogen; aryl,heteroaryl, azido (—N₃), nitro (—NO₂), cyano (—CN), acyloxy(—OC(═O)R′),acyl (—C(═O)R′), alkoxy (—OR′), amido (—NR′C(═O)R″ or —C(═O)NRR′), amino(—NRR′), carboxylic acid (—CO₂H), carboxylic ester (—CO₂R′), carbamoyl(—OC(═O)NR′R″ or —NRC(═O)OR′), hydroxy (—OH), isocyano (—NC), sulfonate(—S(═O)₂OR), sulfonamide (—S(═O)₂NRR′ or —NRS(═O)₂R′), or sulfonyl(—S(═O)₂R), where each R or R′ is selected, independently, from H, C1-C6alkyl or heteroalkyl, C2-C6 alkenyl or heteroalkenyl, 2C-6C alkynyl orheteroalkynyl, aryl, or heteroaryl. A substituted group may have, forexample, 1, 2, 3, 4, 5, 6, 7, 8, or 9 substituents.

By “apheresis,” “hemapheresis,” or “pheresis” is meant the process ofremoving a specific component from the blood, plasma, serum, or afraction thereof, of a subject. Apheresis can be used to remove,separate, or collect one or more specific components of the blood,plasma, serum, or a fraction thereof. In general, apheresis includes thewithdrawal of blood from the subject's body, removal of one or morecomponents from the blood, and transfusion of the remaining blood backinto the subject's body.

By “apolipoprotein L1” or “APOL1” is meant a gene encoding humanapolipoprotein L, 1 (OMIM: 603743; see also SEQ ID NO: 1) or apolypeptide that includes, e.g., amino acids 1-398 of SEQ ID NO: 2.APOL1 is a secreted high density lipoprotein which binds toapolipoprotein A-I. Apolipoprotein A-I is a relatively abundant plasmaprotein and is the major apoprotein of HDL. Several different transcriptvariants encoding different isoforms have been found for this gene.

Nucleic acid and protein sequences for human APOL1 are publiclyavailable. For example, GENBANK® Accession No. NC_000022.10 (nucleotides36649117 . . . 36663577) discloses an exemplary human APOL1 genomicsequence (incorporated by reference as provided by GENBANK® on Apr. 18,2010). In other examples, GENBANK® Accession Nos. AF305224.1,NM_003661.3, NM_145343.2, NM_001136540.1, z82215, and BC127186.1disclose exemplary human APOL1 nucleic acid sequences, and GENBANK®Accession Nos. CAQ09089, NP_003652, AAI43039.1, and AAI42721.1 discloseexemplary human APOL1 protein sequences, all of which are incorporatedby reference as provided by GENBANK® on Apr. 18, 2010.

By “APOL1 risk allele” is meant a form of a gene that is correlated withthe development of, or an increased risk of developing, renal disease ina subject (e.g., a human). The risk allele may correspond to a mutationin an APOL1 gene (e.g., a human APOL1 gene) or a mutation in a gene thatis involved in the expression of a protein that may interacts with anAPOL1 protein or another component of the high density lipoproteincomplex. The mutation may result in a modification (e.g., asubstitution, deletion, or inversion) in a polypeptide product of a riskallele (e.g., a substitution, deletion, or inversion in an APOL1polypeptide). Examples of such mutations in an APOL1 polypeptideinclude, but are not limited to, the G1a, G1b, G1, G2, and G3 mutationsdescribed herein and in PCT/US2011/032924, or combinations thereof. Therisk of renal disease is elevated in subjects carrying one or more(e.g., two, three, or four) APOL1 risk alleles.

By “common APOL1” is meant an APOL1 polypeptide, or nucleic acidmolecule encoding the APOL1 polypeptide, having a sequence that is notcorrelated to the development of renal disease in a subject (e.g., ahuman). For example, a common APOL1 is one that includes an amino acidsequence or a nucleic acid sequence set forth above in the definitionfor “apolipoprotein L1” and “APOL1” (e.g., SEQ ID NOs: 1 and 2), andthat does not include one or more mutations that have been correlatedwith the development of renal disease, such as the G1a, G1b, G1, G2,and/or G3 mutations described herein and in PCT/US2011/032924.

By “decrease” is meant becoming less or smaller, as in number, amount,size, or intensity. In one example, decreasing the risk of a disease(such as FSGS or hypertensive ESKD) includes a decrease in thelikelihood of developing the disease by at least about 20%, for exampleby at least about 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In anotherexample, decreasing the risk of a disease includes a delay in thedevelopment of the disease, for example a delay of at least about sixmonths, such as about one year, such as about two years, about fiveyears, or about ten years.

In one example, decreasing the signs and symptoms of renal disease(e.g., such as FSGS or hypertensive ESKD) includes decreasing theeffects of the disease, such as podocyte injury or glomerular scarringby a desired amount, for example by at least 5%, at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 50%, at least75%, or even at least 90%, as compared to a response in the absence ofadministration of a therapeutic composition of the invention.

By “isolated” (e.g., as an “isolated” biological component, such as anucleic acid molecule, protein, antibody, or cell, or as an “isolated”chemical component, such as a compound or other chemical therapeuticagent) is meant that a component has been substantially separated orpurified away from other biological (or chemical) components, e.g., asin the cell of an organism in which the component naturally occurs. Forexample, a component is “isolated” if it is enriched in a composition,relative to other components in the composition, such that itconstitutes at least 30%, more preferably at least 50%, or even morepreferably at least 75% or more of the composition. A component is“substantially isolated” or “substantially purified” if it is enriched,relative to other components in a composition, such that it constitutesat least 85%, more preferably at least 90%, or even more preferably 95%,97%, or 99% or more of the composition. Nucleic acids and proteins thathave been “isolated” include, e.g., nucleic acid molecules that encodean APOL1 polypeptide or fragment thereof, nucleic acid therapeutics ofthe invention, such an antisense mRNA molecules, protein antagonists ofAPOL1 gene expression, such as those described herein, and antagonistantibodies (e.g., antibodies that target one or more components ofpathways (e.g., those described herein) that are involved in APOL1 geneexpression), each of which may be purified by standard purificationmethods. The term also embraces nucleic acid molecules and proteinsprepared by recombinant expression in a host cell as well as chemicallysynthesized nucleic acid molecules, peptides, and polypeptides (e.g.,one or more of the nucleic acid molecules, proteins, and antibodies ofthe invention).

By “pathogenic APOL1” is meant an APOL1 polypeptide, or nucleic acidmolecule encoding the APOL1 polypeptide, having a sequence that iscorrelated to the development of renal disease in a subject (e.g., ahuman). For example, a pathogenic APOL1 is one that includes an aminoacid sequence or a nucleic acid sequence set forth above in thedefinition for “apolipoprotein L1” and “APOL1” (e.g., SEQ ID NOs: 1 and2), but that also includes one or more mutations that have beencorrelated with the development of renal disease, such as the G1a, G1b,G1, G2, and/or G3 mutations described herein and in PCT/US2011/032924.

By “renal disease” is meant a disorder that specifically leads to damageof the kidneys. Renal diseases include but are not limited to FSGS,hypertensive ESKD, nephropathy secondary to systemic lupuserythematosus, diabetic nephropathy, hypertensive nephropathy, IgAnephropathy, nephritis, HIV-associated nephropathy, non-diabetic chronickidney disease, and xanthine oxidase deficiency.

Renal disease can be chronic or acute. Chronic renal disease, or thetype detected with the assays disclosed herein can progress from stage 1to stage 2, stage 3, stage 4 or stage 5. The stages of chronic renaldisease are:

Stage 1: Slightly diminished kidney function; Kidney damage with normalor increased GFR (>90 mUmin/1.73 m2). Kidney damage is defined aspathologic abnormalities or markers of damage, including abnormalitiesin blood or urine test or imaging studies.

Stage 2: Mild reduction in GFR (60-89 mL/min/1.73 m2) with kidneydamage. Kidney damage is defined as pathologic abnormalities or markersof damage, including abnormalities in blood or urine test or imagingstudies.

Stage 3: Moderate reduction in GFR (30-59 mL/min/1.73 m2)

Stage 4: Severe reduction in GFR (15-29 mUmin/1.73 m2)

Stage 5: Established kidney failure (GFR<15 mL/min/1.73 m2, or permanentrenal replacement therapy (RRT).

The term “pharmaceutically acceptable salt,” as used herein, representsthose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and animalswithout undue toxicity, irritation, allergic response and the like andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge etal. describe pharmaceutically acceptable salts in detail in J. Pharm.Sci. 66:1-19, 1977. The salts can be prepared in situ during the finalisolation and purification of the compounds of the invention orseparately by reacting the free base group with a suitable organic acid.Representative acid addition salts include acetate, adipate, alginate,ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate,butyrate, camphorate, camphersulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate,glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide,hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium, and the like, aswell as nontoxic ammonium, quaternary ammonium, and amine cations,including, but not limited to ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like.

By “specifically binds” is meant the preferential association of abinding moiety (e.g., an antibody, antibody fragment, or antagonist ofan APOL1 gene expression pathway component) to a target molecule (e.g.,a component of a pathway involved in expression of a pathogenic APOL1protein, such as those described herein) in a sample (e.g., a biologicalsample) or in vivo or ex vivo. It is recognized that a certain degree ofnon-specific interaction may occur between a binding moiety and anon-target molecule. Nevertheless, specific binding may be distinguishedas mediated through specific recognition of the target molecule.Specific binding results in a stronger association between the bindingmoiety (e.g., an antibody or fragment thereof) and, e.g., an antigen(e.g., a component of a pathway involved in expression of a pathogenicAPOL1 protein) than between the binding moiety and, e.g., a non-targetmolecule. For example, the antibody may have, e.g., at least 10-foldgreater affinity (e.g., 10, 10²-, 10³-, 10⁴-, 10⁵-, 10⁶-, 10⁷-, 10⁸-,10⁹-, or 10¹⁰-fold greater affinity) to the component of a pathwayinvolved in expression of a pathogenic APOL1 protein than to non-targetprotein.

By “subject” or “patient” is meant a living multi-cellular vertebrateorganism, a category that includes human and non-human mammals (such aslaboratory or veterinary subjects).

By “therapeutically effective amount” is meant an amount of atherapeutic agent (e.g., an agent, such as an antagonist, that targets acomponent of a pathway involved in expression of a pathogenic APOL1protein) that alone, or together with one or more additional (optional)therapeutic agents, induces a desired response. In one example, thedesired response is decreasing the risk of developing FSGS or decreasingthe signs and symptoms of FSGS. For example, a therapeutically effectiveamount of an antagonist of a component of a pathway involved inexpression of a pathogenic APOL1 can be used to treat, prevent, orameliorate renal disease or reduce one or more symptoms associated withrenal disease.

Ideally, a therapeutically effective amount provides a therapeuticeffect without causing a substantial cytotoxic effect in the subject.The preparations disclosed herein are administered in therapeuticallyeffective amounts. In general, a therapeutically effective amount of acomposition administered to a subject (e.g., a human subject) will varydepending upon a number of factors associated with that subject, forexample the overall health of the subject, the condition to be treated,or the severity of the condition. A therapeutically effective amount ofa composition can be determined by varying the dosage of the product andmeasuring the resulting therapeutic response. The therapeuticallyeffective amount can be dependent on the source applied, the subjectbeing treated, the severity and type of the condition being treated, andthe manner of administration.

In one example, a desired response is to prevent the development ofrenal disease (e.g., FSGS). In another example, a desired response is todelay the development or progression of renal disease (e.g., FSGS), forexample, by at least about three months, at least about six months, atleast about one year, at least about two years, at least about fiveyears, or at least about ten years. In another example, a desiredresponse is to decrease the signs and symptoms of renal disease (e.g.,FSGS), such as inflammation and/or scarring of the tissues of thekidney, and/or neurological symptoms in the limbs or associated withspeaking.

By “treatment,” with respect to renal disease, is meant (1) inhibitingdevelopment of symptoms of the disease, e.g., causing the clinicalsymptoms of the disease not to develop in an animal (e.g., a human) thatmay have or be predisposed to develop the disease but does not yetexperience or display symptoms of the disease, (2) inhibiting thedisease, e.g., arresting the development of the disease or one or moreof its clinical symptoms, or (3) relieving or ameliorating the disease,e.g., causing regression of the disease or one or more of its clinicalsymptoms. For example, treatment can refer to relieving one or moresymptoms associated with renal disease. Treatment of a disease does notrequire a total absence of disease. For example, a decrease of at least25% or at least 50% of one or more of the symptoms or undesiredconsequences of the disease can be sufficient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a set of images showing cross-section of mice liver injectedwith WT APOL1 cDNA (left image) or the G1 risk variant APOL1 cDNA(right). The risk variant causes severe and widespread liver injury.

FIG. 2 is a graph showing APOL1 mRNA expression levels in 60 Africans(AA) and Europeans (EA). Expression levels differ widely betweenindividuals.

FIGS. 3A and 3B are graphs showing APOL1 mRNA expression in endothelialcells after stimulation with interferon gamma (FIG. 3A) or interferonbeta (FIG. 3B). Similar effects were observed for endothelial cellstreated with interferon alpha, and for podocytes treated with interferonalpha, beta, or gamma.

FIGS. 4A, 4B, and 4C are graphs showing APOL1 mRNA expression inendothelial cells treated with interferon beta and various Jak kinaseinhibitors: WHI-P131 (FIG. 4A), INCB018424 (FIG. 4B), and TG101348 (FIG.4C). Similar effects were seen in podocytes. Control cells are treatedwith vehicle alone; all other columns represent cells treated withinterferon beta alone or with inhibitor at the indicated concentration.

FIGS. 5A and 5B are graphs showing APOL1 expression in endothelialscells (FIG. 5A) and podocytes (FIG. 5B) after treatment with TLRagonists.

FIGS. 6A and 6B are graphs showing APOL1 expression in endothelial cells(FIG. 6A) and podocytes (FIG. 6B) treated with short, 5′ PPP dsRNA, anagonist of cytoplasmic RIG-L1 receptors.

FIG. 7 is a graph showing APOL1 mRNA expression in endothelial cellstreated with LPS-free bacterial DNA, with or without the transfectionagent Lyovec.

FIGS. 8A, 8B, 8C, and 8D are graphs showing APOL1 mRNA expression inendothelial cells treated with the TLR3 agonist poly(I:C) and differentJAK inhibitors: WHI-131 (FIG. 8A), INCB018424 (FIG. 8B), TG101348 (FIG.8C), CP-690550 (FIG. 8D). Similar effects were seen in podocytes.

FIG. 9 is a graph showing APOL1 expression levels in endothelial cellstreated with TLR3 agonist poly(I:C) and the TBK1lKKe inhibitor Bx795.Similar results were obtained in podocytes. This kinase complex is usedby both TLR and Rig-like receptor signaling mechanisms, and thereforeoffers a broader target for inhibition of APOL1 expression than eitherpathway alone.

FIG. 10 is a graph showing APOL1 expression levels in endothelial cellstreated with TLR3 agonist poly(I:C) and the endosomal acidificationinhibitor chloroquine. Similar results were obtained in podocytes. Thisclass of compounds may be particularly useful for inhibiting APOL1expression when that expression is driven by endosomal patternrecognition receptors such as TLR3, TLR7, TLR8, TLR9, and in some casesTLR4.

FIG. 11 is a graph showing APOL1 expression stimulated by polyl:C wasinhibited by 2 different NF-kB inhibitors. The first inhibitor, Bay11-7085, blocks phosphorylation of lκB, thereby preventing translocationof NF-kB subunits to the nucleus where they regulate gene expression (inthis case APOL1). The second, 6-amino-4-(4-phenoxyphenylethylamino)quinazoline (abbreviated 6A4Q), inhibits NF-kB transcriptionalactivation of target genes. Inhibition at two different steps of theNF-kB activation of APOL1 expression suggests that any of severalhundred other known inhibitors of NF-kB signaling may be useful insuppressing APOL1 expression.

FIG. 12 is a graph showing that an imidazolo-oxindole PKR inhibitor(chemical formula C₁₃H₈N₄OS-EMD chemicals) blocks APOL1 upregulation.

FIG. 13 is a graph showing that a MAP kinase (p38) inhibitor SB203580inhibited APOL1 expression in endothelial cells, consistent withexisting chromatin immunoprecipitation data showing AP-1 binding sitesin the APOL1 promoter.

FIG. 14 is a graph showing that the TLR3 agonist polyl:C stronglystimulates APOL1 expression. Lanes 1 is the control, and lane 2 ispoly(I:C) treated. Virus expressing green fluorescent protein was usedas the viral control in both lanes 1 and 2. All other lanes receivedpolyl:C plus virally delivered shRNA for the specified gene. ReducedAPOL1 expression is seen with knockdown of Jak kinases, Stats, IRFs,NF-kB pathway genes (NEMO, REL-A), and genes involved in TLR signaling(TLR3, Ticam, TBK1).

DETAILED DESCRIPTION

We have discovered that blocking specific signaling pathways causes adecrease in the pathogenic expression of APOL1 polypeptide, thusproviding a method of treatment of renal disease in subjects having aAPOL1 risk variant allele. The invention features agents that targetspecific signaling pathways, e.g., one or more of the JAK/STAT pathway,the TLP pathway, the NF-κB pathway, PKR pathway, the MAP kinase pathway,and the TBK1/lKKe pathway.

About 4 to 5-fold increased risk of non-diabetic end-stage renal diseaseamong African-Americans is caused primarily by mutations in the geneencoding Apolipoprotein L1 (APOL1), a component of the densest, HDL3fraction of high-density lipoprotein. These mutations result in aminoacid deletions, substitutions, and inversions (see, e.g.,PCT/US2011/032924, which is incorporated herein by reference). APOL1variants, commonly found among the subjects of African descent (e.g.,African-Americans and subjects from some regions of Africa), are amongthe most powerful genetic risk factors for any disease yet described interms of frequency and effect size. APOL1 variants can be identified bythe presence of single nucleotide polymorphisms (SNPs) in the APOL1 gene(e.g., a SNP within the C-terminal exon of an APOL1 gene, such as theG1a, G1b, and/or G1 risk alleles), a deletion in the APOL1 gene (e.g.,the G2 risk allele), and/or at least one inversion in an APOL1 gene(e.g., an inversion in a 5′ region of an APOL1 gene, e.g., an inversionin which the 5′ region of an APOL1 gene is replaced with a 5′ region ofan APOL4 gene, such as the G3 risk allele). The first allele, G1, codesfor two amino acid substitutions (S342G and I384M) near the C-terminusthat nearly always occur together (the presence of the first mutationalone, S342G is referred to as the G1a risk allele, while the presenceof the second mutation, I384M, is referred to as the G1b risk allele).The second allele, G2, is a 6 base pair deletion leading to a loss ofamino acid residues 388 and 389 in APOL1. G1 and G2 are mutuallyexclusive, meaning they have not been observed to occur on the samechromosome. Inheritance of two risk alleles (one from each parent) leadsto markedly increased risk of renal disease, whereas one risk allelecauses only a very small increase in risk. G1 and G2 forms of APOL1 wereboth shown to protect against the trypanosomes that cause the deadliestform of African Sleeping Sickness, likely explaining their highfrequency in Africans and African Americans. With allele frequencies inAfrican Americans of 23% and 15%, respectively, G1 and G2 are among themost powerful common risk variants discovered to date. Because G1 and G2are so common in Africans, African Americans, and others of recentAfrican ancestry, we refer to the most common non-risk (wild-type)allele as G0. The major APOL1 haplotypes are shown in Table 1 below.

TABLE Combinations of amino acid variants comprising the major APOL1haplotypes E150K N176S M228I K255R G270D D337N S342G I384M Del388-9 G0(“WT”) E N M R G D S I G1 E N I K G D G M G1a E N I K G D G I G1b E N IK G D S M G2 E N I K G D S I deletion APOL1a K N I K G D S I APOL1b E NI K G N S I APOL1c E S I K G N S I APOL1d E N I K D N S I APOL1e E N I KG D S I

Expression of these risk variants (risk alleles) accounts for most ofthe increased risk of renal disease among African-Americans for severalnon-diabetic kidney diseases, including hypertension-associated kidneydisease, focal segmental glomerulosclerosis, and HIV nephropathy (wherethe increase in risk is nearly 100 fold). A selective advantage of thesepolymorphisms also accounts for their high frequency. Individuals havingthese alleles experience increased protection against African sleepingsickness caused by the parasite Trypanosoma brucei rhodesiense. Whereasprotection against sleeping sickness is a dominant trait, increased riskof renal disease behaves largely as a recessive trait. The compositionsand methods described herein can be used to target these variant APOL1proteins and/or their biological effects. Such an approach can be usedto protect patients with one or more susceptible genotypes against theassociated increased risk of renal disease, e.g., end-stage renaldisease, as well as other patients having or likely to develop renaldisease.

APOL1 has evolved recently under high selective pressure and is foundonly in humans and a few other primate species. APOL1 has beenextensively studied as a component of trypanolytic factor 1 (TLF-I), butalmost nothing else is known about APOL1 biology in higher primates. Thefew published papers document the presence of APOLI in HDL, but itsfunction in this complex is unknown.

APOL1 variants increase risk of renal disease from multiple etiologies.Expression of two or more risk-associated APOL1 variants increasesdisease risk between 10- and 100-fold. For example, the risk increase isapproximately 7 to 10-fold in H-ESRD, 10-17-fold in FSGS, and 30-foldfor HIV. Since the same variants have such profound effects on distincttypes of kidney disease, similar pathogenic mechanisms may underlie theenhanced risk for all these entities, if not the disease mechanismsthemselves. We believe this increase in risk represents one of thelargest ever attributed to common variants. In this context we suggestthat APOL1-related kidney disease might be better characterized as aMendelian disease with modifiers, rather than a common, complex disease.

Nephrologists have little to offer to their patients to slow theprogression of diagnosed renal diseases, such as FSGS and ESKD. Currenttreatments include, e.g., angiotensin-converting enzyme inhibitors(ACEi) and angiotensin II receptor blockers (ARBs), but these treatmentsare non-specific and only moderately effective. Very few patientsrespond to steroids, and many of those only transiently.

The APOL1 protein product is a secreted lipoprotein with homology toBcl-2 family members (Duchateau et al., J. Biol. Chem. 272:25576-25582,1997; and Wan et al., J. Biol. Chem. 283:21540-21549, 2008). Itcirculates as part of HDL3 complexes, the densest HDL fraction, and isexpressed in multiple tissue types. It has putative roles in autophagyand apoptosis, and plays an important role in the innate immune systemas the human trypanolytic factor that protects humans and some otherprimates from parasites of the trypanosomida family. The renal riskvariants prevent the C-terminus of APOL1 from binding to the trypanosomevirulence factor SRA. Evolution of the SRA factor to bind the leucinezipper of APOL1 suggests the existence of an endogenous binding partnerfor APOL1 that may be critical in governing its function.

APOL1's BH3-only domain strongly suggests a role in apoptosis. BH3-onlyproteins are pro-apoptotic because they antagonize anti-apoptotic Bcl2family members with BH3-binding grooves such as Bcl2 itself (Adams etal., Curr. Opin. Immunol. 19:488-496, 2007). In our studies, we observedfrequent cell death in oocytes injected with APOL1. However, when weco-injected Bcl-XL (a Bcl-2-like anti-apoptotic protein) together withAPOL1, oocyte death was essentially eliminated. This finding suggeststhat APOL1 causes apoptotic cell death via its BH3 domain.

We have discovered that agents (e.g., agents that target specificsignaling pathways such as the the JAK/STAT pathway, the TLP pathway,the NF-κB pathway, PKR pathway, the MAP kinase pathway, and theTBK1/lKKe pathway) that decrease the deleterious effects of pathogenicAPOL1 high risk variants provide a beneficial therapy for patientshaving or that will progress to end-stage renal disease, as well asthose patients that will likely require renal replacement therapy.

Susceptibility to kidney disease associated with the high-risk variantsof APOL1 behaves like a recessive trait (Genovese et al., Science329:841-845, 2010). For example, we observed that a human (age 51) withhomozygous null APOL1 mutations that was identified after infection withthe opportunistic pathogen Trypanosoma evansii displayed no evidence ofeither renal dysfunction or additional immune dysfunction. This suggestsa gain-of-function phenotype of APOL1 renal risk variants despiterecessive inheritance. In addition, heterozygotes appear not to developrenal disease, suggesting that the wild-type protein variant protectsagainst coexpressed renal risk variants in APOL1 heterozygotes.Furthermore, the presence of two high-risk APOL1 alleles confersapproximately 10-fold increased risk of FSGS and (in the context of HIVinfection) approximately 100-fold increased risk of HIV nephropathy.Also, HDL3, which contains APOL1, appears to be filtered by theglomerulus (Breznan et al., Biochem. J. 379:343-349, 2004), and APOL1protein is immunodetected at high levels in proximal tubular epithelialcells, likely reflecting endocytic uptake of filtered HDL3, as well asin the glomerulus and renal vasculature. Among the six human APOL geneproducts, APOL1 is the only secreted protein.

Circulating APOL1 acts on trypanosomes via trypanosome receptor uptakeand subsequent trafficking through the endosomal pathway to thelysosome. The high risk variants of APOL1 are similarly endocytosed fromglomerular filtrate and the interstitial fluid by the kidney, and theseprocesses appear to be required for accelerated progression of renaldisease in affected individuals. In view of these observations, theinvention provides methods for targeting various signaling pathways todecrease the expression level of the pathogenic APOL1 variants inpatients having or likely to develop renal disease, which thereby treatsor reduces the symptoms or likelihood of developing renal disease inpatients at risk.

Methods of the Invention

The methods of the invention include the administration of one or moreagents that target one or more signaling pathways (e.g., one or morecomponents of the JAK/STAT pathway, the TLP pathway, the NF-κB pathway,PKR pathway, the MAP kinase pathway, and the TBK1/lKKe pathway) in cellsof a patient, thereby decreasing the expression levels of a pathogenicAPOL1 polypeptide, which is encoded by an APOL1 risk allele in thepatient. The agents of the invention can be administered to treat orprevent renal disease (e.g., FSGS, ESKD or non-diabetic chronic kidneydisease) or to ameliorate or reduce one or more symptoms of renaldisease, in patients in need thereof (e.g., patients having one or moreAPOL1 risk alleles). The invention features methods of treating asubject (e.g., a human subject) having or at risk of developing a renaldisease.

One or more of the agents of the invention can also be administered to asubject in need of kidney transplantation (e.g., prior to or aftertransplantation). In a preferred embodiment, the subject is one that hasbeen found to have at least one APOL1 gene risk allele (e.g., a G1a,G1b, G1, G2, and/or G3 mutation). It is known that individuals ofAfrican ancestry, including those individuals of Hispanic ancestry and,in particular, African-Americans, have an elevated risk for carrying oneor two copies of at least one risk allele of the APOL1 gene, whichincreases their risk of developing idiopathic kidney disease. Thus, inone embodiment, a kidney recipient can be genotyped to determine if therecipient carries one, two, or more copies of at least one of thedisclosed risk alleles of the APOL1 gene and can be treated prior to orafter kidney transplantation with one or more of the agents of theinvention. Additionally, a kidney selected for transplantation can betreated with one or more of the compositions of the invention prior totransplantation of the kidney into the recipient.

The agents of the invention can target any one or more components of theJAK/STAT pathway, the TLP pathway, the NF-κB pathway, PKR pathway, theMAP kinase pathway, and the TBK1/lKKe pathway. To monitor the efficacyof the treatment, methods for assaying levels of APOL1 expression afteradministration of an agent of the invention (e.g., an agent targetingany one or more components of the JAK/STAT pathway, the TLP pathway, theNF-κB pathway, PKR pathway, the MAP kinase pathway, and the TBK1/lKKepathway) can be used. These methods can include, e.g., western blotting,immunoprecipitation, ELISA, and q-RT-PCR.

Agents that Target the Janus Kinase (JAK)/Signal TransductionTranscriptional Activation (STAT) Pathway

The methods of the invention feature the administration of agents (e.g.,antagonists) that target one or more components of the JAK/STAT pathway,thereby decreasing the levels of APOL1 polypeptide expression (e.g., apathogenic APOL1) in a cell. Administration of the agent(s) treats,prevents, reduces, ameliorates, or alleviates one or more symptoms of arenal disease (e.g., FSGS, ESKD, or non-diabetic chronic kidneydisease). The agent(s) target can target any one or more of JAK1, JAK2,JAK3, and TYK2. For example, these agents can be antagonists of JAKsthat decrease pathogenic APOL1 polypeptide expression caused byinflammatory factors. The JAK antagonists can be any one or more oflestaurtinib, CP-690550 (tofacitinib), ruxolitinib, SB1518 (pacritinib),CYT387, LY3009104, INCB28050 (baricitinib), TG101348, SD1008,cucrbitacin, G06976, WHI-P154, and AG490.

JAK inhibitors that may be used in the present invention are describedin U.S. patent application publication Nos. 20100113416, 20070135461,20060106020, 20060183906, 20070149506, 20080188500, and in U.S. Pat.Nos. 7,705,004; 8,258,144; and 8,138,339; and 6,825,190 (each of whichis incorporated herein by reference).

The agents can target specific JAKs, or may target more than one JAKsimultaneously. For example, lestaurtinib targets JAK2, CP-690550(tofacitinib) targets JAK3, ruxolitinib targets JAK1 and JAK2, SB1518(pacritinib) targets JAK2, CYT387 targets JAK2, LY3009104 and INCB28050(baricitinib) targets JAK1 and JAK2, TG101348 targets JAK2, SD1008targets JAK2, cucrbitacin targets JAK2, WHI-P154 targets JAK3, and AG490targets JAK2.

The agent can also target STAT1, STAT2, STAT3, STAT4, STAT5, and/orSTAT6. STAT inhibitors that may be used in the methods of the inventionare described in U.S. Pat. Nos. 6,884,782 and 8,143,412 (each of whichis incorporated herein by reference).

The agents can target specific STATs, or may target more than one STATsimultaneously and can be any one or more of WP1066 (targets STAT3);WP1064 (targets STAT3 and STAT5); STA21; STAT3 Inhibitor V, Stattic;STAT3 Inhibitor VI, S3I-201; STAT3 Inhibitor VII; cucurbitacin (targetsSTAT3); SD1008 (targets STAT3) and STAT3 Inhibitor VIII, 5,15-DPP.

In some embodiments of the above aspects, the agents may also target oneor more JAK and/or STAT targets simultaneously.

Agents that Target the Toll-Like Receptor (TLR) Pathway

The methods of the invention also feature the administration of one ormore agents (e.g., antagonists) that target one or more components ofthe TLR pathway, thereby decreasing the levels of APOL1 polypeptideexpression (e.g., a pathogenic APOL1) in a cell. Administration of theagent(s) treats, prevents, reduces, ameliorates, or alleviates one ormore symptoms of a renal disease (e.g., FSGS, ESKD, or non-diabeticchronic kidney disease). These agents target can target any one or moreof TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11,TLR12, and TLR13. For example, these agents can be antagonists of theTLRs that decrease pathogenic APOL1 polypeptide expression caused byinflammatory factors. The TLR antagonists can be any one or more oferitoran, naloxone, naltrexone, LPS-RS, ibudilast, propentofulline,amitriptyline, ketotifen, cyclobenzaprine, mianserin, imipramine,RSCL-0518, RSCL-0519, RSCL-0575, RSCL-0521, RSCL-0520, RSCL-0638, andRSCL-0522, eritoran, chloroquine, and an oligonucleotide-basedantagonist. Oligonucleotide-based TLR antagonists include one or more ofthose described in e.g., U.S. Patent Application Publication No.20090060898 (incorporated herein by reference). Additional TLRantagonists that may be used in the methods of the invention aredescribed in U.S. Patent Application Publication No. 20100098685(incorporated herein by reference).

Agents that Target the NF-κB Pathway

The methods of the invention feature the administration of one or moreagents (e.g., an antagonist) that target the NF-κB pathway, therebydecreasing the levels of APOL1 polypeptide expression (e.g., apathogenic APOL1) in a cell. Administration of the agent(s) treats,prevents, reduces, ameliorates, or alleviates one or more symptoms of arenal disease (e.g., FSGS, ESKD, or non-diabetic chronic kidneydisease). For example, these agents can be antagonists of NF-κB thatdecrease pathogenic APOL1 polypeptide expression caused by inflammatoryfactors. The NF-κB antagonists can be any one or more of Disulfiram,olmesartan, dithiocarbamates, 2-(1,8-naphthyridin-2-yl)-Phenol,5-Aminosalicylic acid, BAY 11-7082, BAY 11-7085, CAPE (Caffeic AcidPhenethylester), Diethylmaleate, lKK-2 Inhibitor IV, IMD0354,lactacystin, MG-132 [Z-Leu-Leu-Leu-CHO], NFκB Activation Inhibitor III,NF-κB Activation Inhibitor II, JSH-23, parthenolide, phenylarsine oxide,PPM-18, pyrrolidinedithiocarbamic acid ammonium salt, QNZ, RO 106-9920,rocaglamide, rocaglamide AL, rocaglamide C, rocaglamide I, rocaglamideJ, rocaglaol, (R)-MF-132, sodium salicylate, triptolide (PG490), 6A4Q,capsaicin, andrographolide, aurothiomalate, (5Z)-7-oxozeanol,evodiamine, helenalin, gliotoxin, hypoestoxide, NF-κB inhibitor, NE-κBinhibitor V 5HPP-33, NE-κB inhibitor VI BOT-64, NE-κB inhibitor VIICID-2858522, NF-κB inhibitor VIII A-UBI, SN50, SN50M, NF-κB inhibitorVIII EVP4593, nodinitib, oridonin, PPM-18, parthenolide, sulfasalazine,ursolic acid, TIRAP inhibitor peptide, and wedelolactone. AdditionalNF-κB inhibitors that may be used in the methods of the invention aredescribed e.g., in U.S. Pat. No. 6,410,516 (incorporated herein byreference).

Agents that Target the MAP Kinase Pathway

The methods of the invention feature the administration of one or moreagents (e.g., antagonists) that target one or more components of the MAPkinase pathway, thereby decreasing the levels of APOL1 polypeptideexpression (e.g., a pathogenic APOL1) in a cell. Administration of theagent(s) treats, prevents, reduces, ameliorates, or alleviates one ormore symptoms of a renal disease (e.g., FSGS, ESKD, or non-diabeticchronic kidney disease). For example, these agents can be antagonists ofthe p38 MAP kinase that decrease pathogenic APOL1 polypeptide expressioncaused by inflammatory factors. The MAP kinase antagonists can be anyone or more of CAY10571, JX-401, MK2a inhibitor, p38 MAP kinaseinhibitor, p38 MAP kinase inhibitor III, p38 MAP kinase inhibitor IV,CGH 2466, p38 MAP kinase inhibitor V, PD169316, SB202190, SB203580,SB239063, SB220025, SD-169, RWJ 67657, Antibiotic LL Z1640-2, SCIO 469hydrochloride, Tie2 kinase inhibitor, VX 475, p38 MAP kinase inhibitorIX, SX 011, TAK 715, VX 702, SB 202190 hydrochloride, ZM 336372, SKF86002, pamapimod, PF-797804, SB203580, SP600125, SB202190, and SB239906,and doramapimod. Additional MAP kinase antagonists that may be used inthe methods of the invention are described, for example, in U.S. PatentApplication Publication No. 20120129867 (incorporated herein byreference).

Agents that Target the Protein Kinase R (PKR) Pathway

The methods of the invention feature the administration of one or moreagents (e.g., antagonists) that target one or more components of the PKRpathway, thereby decreasing the levels of APOL1 polypeptide expression(e.g., a pathogenic APOL1) in a cell. Administration of the agent(s)treats, prevents, reduces, ameliorates, or alleviates one or moresymptoms of a renal disease (e.g., FSGS, ESKD, or non-diabetic chronickidney disease). For example, these agents can be antagonists of theprotein kinase R (PKR) that decrease pathogenic APOL1 polypeptideexpression caused by inflammatory factors. The PKR antagonist can have ageneral structure according to the following formula (I),

or a stereoisomer thereof, or a pharmaceutically acceptable saltthereof, wherein each of R¹ and R² is, independently, selected from H,optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy,halogen, or CN; each of X¹ and X² is, independently, selected from O, S,NR^(X), or C(R^(X))₂, where each R^(X) is, independently, H oroptionally substituted C1-C6 alkyl, or one R^(X) can combine with R³ toform a double bond; R³ is H, optionally substituted C1-C6 alkyl, orhalogen, or R³ combines with one R^(X) to form a double bond; R⁴ is H,or R⁴ combines with R⁵ to form a double bond, or R⁴ combines with R⁶ toform a double bond; R⁵ is H or optionally substituted C1-C6 alkyl, or R⁵combines with R⁴ to form a double bond; each of R⁶ and R⁷ is,independently, H or optionally substituted C1-C6 alkyl, or R⁶ combineswith R⁴ to form a double bond, or R⁶ and R⁷ combine to form an oxo (═O)or a thioxo (═S) group; R⁸ is H or optionally substituted C1-C6 alkyl;and Ar is an optionally substituted phenyl or an optionally substitutedfive- or six-membered heteroaryl.

The antagonist can have the structure according to formula (I-A),

or a stereoisomer thereof, or a pharmaceutically acceptable saltthereof, wherein X⁴ is O or S; X⁵ is O, S, or NR^(X); and X⁶ is N or CH.

The PKR antagonist can also have the structure

Alternatively, the PKR antagonist can be 2-aminopurine or P581PK.Agents that Target the TANK-Binding Kinase (TBK) 1/lκB Kinase e (lKKe)Pathway

The methods of the invention feature the administration of one or moreagents (e.g., antagonists) that target one or more components of theTBK1/lKKe pathway, thereby decreasing the levels of APOL1 polypeptideexpression (e.g., a pathogenic APOL1) in a cell. Administration of theagent(s) treats, prevents, reduces, ameliorates, or alleviates one ormore symptoms of a renal disease (e.g., FSGS, ESKD, or non-diabeticchronic kidney disease). For example, these agents can be antagonists ofthe TBK1/lKKe pathway that decrease pathogenic APOL1 polypeptideexpression caused by inflammatory factors. The TBK1/lKKe antagonists canbe any one or more of Bx795, auranofinc, PS-1145 dihydrochloride,wedelolactone, lKK inhibitor X, butein, and lKK16, The TBK1/lKKeantagonist can also be any one or more of binding proteins or bindingpeptides directed against TBK-1, in particular against the active siteof TBK-1, and nucleic acids directed against the TBK-1 gene. Preferably,the inhibitor binds to the ATP-binding site of the kinase domain ofTBK-1. The TBK1/lKKe antagonists can be selected from the groupconsisting of antisense oligonucleotides, antisense RNA, siRNA, and lowmolecular weight molecules (LMWs), which are not proteins, peptidesantibodies or nucleic acids, and which exhibit a molecular weight ofless than 5000 Da, preferably less than 2000 Da, more preferably lessthan 2000 Da, most preferably less than 500 Da as described in U.S.Patent Application Publication No.: 20070149469.

Therapeutic RNA Interference Agents of the Invention that TargetExpression of APOL1

The present invention also features the administration of one or moretherapeutic ribonucleic acid interference agents (RNAi) that can be usedto decrease the levels of APOL1 polypeptide expression (e.g., apathogenic APOL1) in a cell for the treatment of disease (e.g., a renaldisease). For example, the agents may be RNAi agents that target one ormore components of the JAK/STAT pathway, the TLP pathway, the NF-κBpathway, PKR pathway, the MAP kinase pathway, and/or the TBK1/lKKepathway, thereby decreasing the levels of APOL1 polypeptide expression(e.g., a pathogenic APOL1) in a cell. Administration of one or more ofthese RNAi agents treats, prevents, reduces, ameliorates, or alleviatesone or more symptoms of a renal disease (e.g., FSGS, ESKD, ornon-diabetic chronic kidney disease).

Such therapeutic RNAi agents include, e.g., antisense nucleobaseoligomers, microRNAs, dsRNA, or small interfering RNAs that downregulateexpression of APOL1 mRNA directly.

The RNAi agents can decrease pathogenic APOL1 polypeptide expression bydownregulating the expression of one or more genes selected form thegroup consisting of JAK1, JAK2, JAK3, STAT1, STAT2, STAT3, STAT4,STAT5A, STAT6, NEMO, REL-1, TLR3, TICAM1, TBK1, IRF1, IRF2, IRF3, andIRF9.

Methods for assaying levels of APOL1 protein expression after RNAinterference are also well known in the art and include, e.g., Westernblotting, immunoprecipitation, and ELISA.

RNA Interference Agents

While the first described RNAi molecules were RNA: RNA hybridscomprising both an RNA sense and an RNA antisense strand, it has nowbeen demonstrated that DNA sense:RNA antisense hybrids, RNA sense:DNAantisense hybrids, and DNA:DNA hybrids are capable of mediating RNAi(Lamberton et al., Molec. Biotechnol. 24:111-119, 2003). Thus, theinvention includes the use of RNAi molecules comprising any of thesedifferent types of double-stranded molecules, which can be used toreduce expression of a pathogenic APOL1. In addition, it is understoodthat RNAi molecules may be used and introduced to cells in a variety offorms. Accordingly, as used herein, RNAi molecules encompasses any andall molecules capable of inducing an RNAi response in cells, including,but not limited to, double-stranded polynucleotides comprising twoseparate strands, i.e. a sense strand and an antisense strand, e.g.,small interfering RNA (siRNA); polynucleotides comprising a hairpin loopof complementary sequences, which forms a double-stranded region, e.g.,shRNAi molecules, and expression vectors that express one or morepolynucleotides capable of forming a double-stranded polynucleotidealone or in combination with another polynucleotide.

RNA interference (RNAi) may be used to specifically inhibit expressionof target polynucleotides (e.g., one or more genes selected form thegroup consisting of JAK1, JAK2, JAK3, STAT1, STAT2, STAT3, STAT4,STAT5A, STAT6, NEMO, REL-1, TLR3, TICAM1, TBK1, IRF1, IRF2, IRF3, andIRF9). Double-stranded RNA-mediated suppression of gene and nucleic acidexpression may be accomplished according to the invention by introducingdsRNA, siRNA or shRNA into cells or organisms. SiRNA may bedouble-stranded RNA, or a hybrid molecule comprising both RNA and DNA,e.g., one RNA strand and one DNA strand. It has been demonstrated thatthe direct introduction of siRNAs to a cell can trigger RNAi inmammalian cells (Elshabir et al., Nature 411:494-498, 2001).Furthermore, suppression in mammalian cells occurred at the RNA leveland was specific for the targeted genes, with a strong correlationbetween RNA and protein suppression (Caplen et al., Proc. Natl. Acad.Sci. USA 98:9746-9747, 2001). In addition, it was shown that a widevariety of cell lines, including HeLa S3, COS7, 293, NIH/3T3, A549,HT-29, CHO-KI and MCF-7 cells, are susceptible to some level of siRNAsilencing (Brown et al., TechNotes 9(1):1-7, 2002).

Exemplary RNAi agents include siRNA, shRNA, dsRNA, and miRNA agents. Incertain embodiments, the RNAi agent is a small interfering RNA (siRNA).These are are short (usually 21 nt) and are usually double-stranded RNA(dsRNA). siRNA molecules may have, for example, 1 or 2 nucleotideoverhangs on the 3′ ends, or may be blunt-ended. Each strand has a 5′phosphate group and a 3′ hydroxyl group. Most siRNA molecules are 18 to30 (e.g., 21 to 30) nucleotides in length, however a skilledpractitioner may vary this sequence length (e.g., to increase ordecrease the overall level of gene silencing).

Almost any gene for which the sequence is known can thus be targetedbased on sequence complementarity with an appropriately tailored siRNA.See, for example, Zamore et al., Cell 101:25-33, 2000; Bass, Nature411:428-429, 2001; Elbashir et al., Nature 411:494-498, 2001; and PCTPublication Nos. WO 00/44895, WO 01/36646, WO 99/32619, WO 00/01846, WO01/29058, WO 99/07409, and WO 00/44914. Methods for preparing a siRNAmolecule are known in the art and described in, for example, U.S. Pat.No. 7,078,196. Accordingly, one of skill in the art would understandthat a wide variety of different siRNA molecules may be used to target aspecific gene or transcript. In certain embodiments, siRNA moleculesaccording to the invention are double-stranded and 16-30 or 18-25nucleotides in length, including each integer in between. In oneembodiment, an siRNA is 21 nucleotides in length. In certainembodiments, siRNAs have 0-7 nucleotide 3′ overhangs or 0-4 nucleotide5′ overhangs. In one embodiment, a siRNA molecule has a two nucleotide3′ overhang. In one embodiment, a siRNA is 21 nucleotides in length withtwo nucleotide 3′ overhangs (e.g., they contain a 19 nucleotidecomplementary region between the sense and antisense strands). Incertain embodiments, the overhangs are UU or dTdT 3′ overhangs.

Generally, siRNA molecules are completely complementary to one strand ofa target DNA molecule, since even single base pair mismatches have beenshown to reduce silencing. In other embodiments, siRNAs may have amodified backbone composition, such as, for example, 2′-deoxy- or2′-O-methyl modifications. However, in preferred embodiments, the entirestrand of the siRNA is not made with either 2′ deoxy or 2′-O-modifiedbases. In one embodiment, siRNA target sites are selected by scanningthe target mRNA transcript sequence for the occurrence of AAdinucleotide sequences. Each AA dinucleotide sequence in combinationwith the 3′ adjacent approximately 19 nucleotides are potential siRNAtarget sites. In one embodiment, siRNA target sites are preferentiallynot located within the 5′ and 3′ untranslated regions (UTRs) or regionsnear the start codon (within approximately 75 bases), since proteinsthat bind regulatory regions may interfere with the binding of the siRNPendonuclease complex (Elshabir et al., Nature 411:494-498, 2001;Elshabir et al., EMBO J. 20:6877-6888, 2001). In addition, potentialtarget sites may be compared to an appropriate genome database, such asBLASTN 2.0.5, and potential target sequences with significant homologyto other coding sequences eliminated.

A short hairpin RNA (shRNA) molecule may also be used in the methods ofthe invention to target one or more components of the JAK/STAT pathway,the TLP pathway, the NF-κB pathway, PKR pathway, the MAP kinase pathway,and/or the TBK1/lKKe pathway. shRNA are single-stranded RNA molecules inwhich a tight hairpin loop structure is present, allowing complementarynucleotides within the same strand to form bonds. shRNA can exhibitreduced sensitivity to nuclease degradation as compared to siRNA. Onceinside a target cell, shRNA are processed and effect gene silencing bythe same mechanism described above for siRNA. In certain embodiments,they may contain variable stem lengths, typically from 19 to 29nucleotides in length, or any number in between. In certain embodiments,hairpins contain 19 to 21 nucleotide stems, while in other embodiments,hairpins contain 27 to 29 nucleotide stems. In certain embodiments, loopsize is between 4 to 23 nucleotides in length, although the loop sizemay be larger than 23 nucleotides without significantly affectingsilencing activity. shRNA molecules may contain mismatches, for exampleG-U mismatches between the two strands of the shRNA stem withoutdecreasing potency. In certain embodiments, shRNAs are designed toinclude one or several G-U pairings in the hairpin stem to stabilizehairpins during propagation in bacteria, for example. However,complementarity between the portion of the stem that binds to the targetmRNA (antisense strand) and the mRNA is typically required, and even asingle base pair mismatch is this region may abolish silencing. 5′ and3′ overhangs are not required, since they do not appear to be criticalfor shRNA function, although they may be present (Paddison et al., Genes& Dev. 16(8):948-958, 2002).

Double-stranded RNA (dsRNA) can also be used in the methods of theinvention to target one or more components of the JAK/STAT pathway, theTLP pathway, the NF-κB pathway, PKR pathway, the MAP kinase pathway,and/or the TBK1/lKKe pathway. Any double-stranded RNA that can becleaved in cell into siRNA molecules that target a specific mRNA can beused. Methods of preparing dsRNA for use as RNAi agents are describedin, for example, U.S. Pat. No. 7,056,704.

MicroRNAs (miRNA) can also be used in the invention. miRNA aresingle-stranded RNA molecules that can silence a target gene (e.g., agene encoding a protein that is a component involved in one or more ofthe JAK/STAT pathway, the TLP pathway, the NE-κB pathway, PKR pathway,the MAP kinase pathway, and/or the TBK1/lKKe pathway) using the same orsimilar mechanisms as siRNA and shRNA agents. miRNA molecules of 17 to25 (e.g., 21 to 23) nucleotides in length are often used, as these aregenerally the most effective for gene silencing; however, a skilledpractitioner may vary the sequence length as desired.

The nucleic acid can be an antisense oligonucleotide directed to atarget polynucleotide. Antisense oligonucleotides are single strands ofDNA or RNA that are complementary to a chosen sequence. In the case ofantisense RNA, they prevent translation of complementary RNA strands bybinding to it. Antisense DNA can be used to target a specific,complementary (coding or non-coding) RNA. If binding takes places thisDNA/RNA hybrid can be degraded by the enzyme RNase H. In a particularembodiment, antisense oligonucleotides contain from about 10 to about 50nucleotides, more preferably about 15 to about 30 nucleotides. The termalso encompasses antisense oligonucleotides that may not be exactlycomplementary to the desired target gene. Thus, antisense agents can beutilized in methods of the invention in instances where non-targetspecific-activities are found with antisense, or where an antisensesequence containing one or more mismatches with the target sequence isthe most preferred for a particular use.

Antisense oligonucleotides have been demonstrated to be effective andtargeted inhibitors of protein synthesis, and, consequently, can be usedto specifically inhibit protein synthesis by a targeted gene (e.g., apathogenic APOL1 polypeptide, but preferably not a common APOL1polypeptide or one or more genes encoding protein components involved inthe JAK/STAT pathway, the TLP pathway, the NF-κB pathway, PKR pathway,the MAP kinase pathway, and/or the TBK1/lKKe pathway). The efficacy ofantisense oligonucleotides for inhibiting protein synthesis is wellestablished. For example, the synthesis of polygalactauronase and themuscarine type 2 acetylcholine receptor are inhibited by antisenseoligonucleotides directed to their respective mRNA sequences (U.S. Pat.Nos. 5,739,119 and 5,759,829). Further, examples of antisense inhibitionhave been demonstrated with the nuclear protein cyclin, the multipledrug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABAAreceptor and human EGF (see e.g., Jaskulski et al., Science240(4858):1544-1546, 1988; Vasanthakumar et al., Cancer Commun.1(4):225-232, 1989; Peris et al., Brain Res Mol Brain Res. 57(2):310-20,1998; and U.S. Pat. Nos. 5,801,154; 5,789,573; 5,718,709; and5,610,288). Furthermore, antisense constructs have also been describedthat inhibit and can be used to treat a variety of abnormal cellularproliferations, e.g. cancer (U.S. Pat. Nos. 5,747,470; 5,591,317; and5,783,683).

Methods of producing antisense oligonucleotides are known in the art andcan be readily adapted to produce an antisense oligonucleotide thattargets any polynucleotide sequence. Selection of antisenseoligonucleotide sequences specific for a given target sequence is basedupon analysis of the chosen target sequence and determination ofsecondary structure, Tm, binding energy, and relative stability.Antisense oligonucleotides may be selected based upon their relativeinability to form dimers, hairpins, or other secondary structures thatwould reduce or prohibit specific binding to the target mRNA in a hostcell. Highly preferred target regions of the mRNA include those regionsat or near the AUG translation initiation codon and those sequences thatare substantially complementary to 5′ regions of the mRNA. Thesesecondary structure analyses and target site selection considerationscan be performed, for example, using v.4 of the OLIGO primer analysissoftware (Molecular Biology Insights) and/or the BLASTN 2.0.5 algorithmsoftware (Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997).

According to another embodiment of the invention, nucleic acid-lipidparticles can be associated with ribozymes. Ribozymes are RNA-proteincomplexes having specific catalytic domains that possess endonucleaseactivity (Kim et al., Proc Natl Acad Sci USA 84(24):8788-8792, 1987;Forster et al., Cell 49(2):211-220, 1987). For example, a large numberof ribozymes accelerate phosphoester transfer reactions with a highdegree of specificity, often cleaving only one of several phosphoestersin an oligonucleotide substrate. This specificity has been attributed tothe requirement that the substrate bind via specific base-pairinginteractions to the internal guide sequence (“IGS”) of the ribozymeprior to chemical reaction. The enzymatic nucleic acid molecule may beformed in a hammerhead, hairpin, a hepatitis δ virus, group I intron orRNaseP RNA (in association with an RNA guide sequence) or Neurospora VSRNA motif, for example. Specific examples of hammerhead motifs aredescribed by Rossi et al., Nucleic Acids Res. 20(17):4559-4565, 1992.Examples of hairpin motifs are described by Hampel et al. (Eur. Pat.Appl. Publ. No. EP 0360257), Hampel et al., Biochemistry28(12):4929-4933, 1989; Hampel et al., Nucleic Acids Res. 18(2):299-304,1990, and U.S. Pat. No. 5,631,359; and an example of the Group I intronis described in U.S. Pat. No. 4,987,071. Ribozymes may be designed asdescribed in Int. Pat. Appl. Publ. No. WO 93/23569 and Int. Pat. Appl.Publ. No. WO 94/02595, each specifically incorporated herein byreference, and synthesized to be tested in vitro and in vivo, asdescribed therein. Ribozyme activity can be optimized by altering thelength of the ribozyme binding arms or chemically synthesizing ribozymeswith modifications that prevent their degradation by serum ribonucleases(see e.g., Int. Pat. Appl. Publ. No. WO 92/07065; Int. Pat. Appl. Publ.No. WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl.Publ. No. 92110298.4; U.S. Pat. No. 5,334,711; and Int. Pat. Appl. Publ.No. WO 94/13688, which describe various chemical modifications that canbe made to the sugar moieties of enzymatic RNA molecules), modificationswhich enhance their efficacy in cells, and removal of stem loop bases toshorten RNA synthesis times and reduce chemical requirements.

A variety of methods are available for the introduction (e.g.,transfection) of nucleic acid molecules (e.g., RNAi agents) intomammalian cells. For example, there are several commercially-availabletransfection reagents useful for lipid-based transfection of siRNAsincluding, but not limited to, TransIT-TKO™ (Mirus, Catalog No. MIR2150), Transmessenger™ (Qiagen, Catalog No. 301525), Oligofectamine™ andLipofectamine™ (Invitrogen, Catalog No. MIR 12252-011 and Catalog No.13778-075), siPORT™ (Ambion, Catalog No. 1631), and DharmaFECT™ (FisherScientific, Catalog No. T-2001-01). Agents are also commerciallyavailable for electroporation-based methods for transfection of siRNA,such as siPORTer™ (Ambion, Catalog No. 1629). Microinjection techniquesmay also be used. The nucleic acid molecule may also be transcribed froman expression construct introduced into the cells, where the expressionconstruct includes a coding sequence for transcribing the nucleic acidmolecule operably linked to one or more transcriptional regulatorysequences. Where desired, plasmids, vectors, or viral vectors can alsobe used for the delivery of the nucleic acid molecule, and such vectorsare known in the art. Additional methods are known in the art and aredescribed, for example, in U.S. Patent Application Publication No.2006/0058255.

Any of the RNAi molecules described herein may be modified orsubstituted with nucleotide analogs, e.g., as described herein. RNAiagents may be capable of silencing any gene where a reduction inexpression of that gene is therapeutically beneficial, e.g., byresulting in a reduction of APOL1 polypeptide expression, or in theexpression of a gene encoding a component involved in the JAK/STATpathway, the TLP pathway, the NF-κB pathway, PKR pathway, the MAP kinasepathway, and/or the TBK1/lKKe pathway, the silencing of which mediates adecrease in expression of a pathogenic APOL1 polypeptide.

Modified Nucleic Acids for Use in the RNAi Molecules of the Invention

Modified nucleic acids, including modified DNA or RNA molecules, may beused in the in place of naturally occurring nucleic acids in the RNAipolynucleotides described herein. Modified nucleic acids can improve thehalf-life, stability, specificity, delivery, solubility, and nucleaseresistance of the polynucleotides described herein. For example, siRNAagents can be partially or completed composed of nucleotide analogs thatconfer the beneficial qualities described above. As described in Elménet al. (Nucleic Acids Res. 33:439-447, 2005), synthetic, RNA-likenucleotide analogs (e.g., locked nucleic acids (LNA)) can be used toconstruct siRNA molecules that exhibit silencing activity against atarget gene product.

Modified nucleic acids include molecules in which one or more of thecomponents of the nucleic acid, namely sugars, bases, and phosphatemoieties, are different from that which occurs in nature, preferablydifferent from that which occurs in the human body. Nucleosidesurrogates are molecules in which the ribophosphate backbone is replacedwith a non-ribophosphate construct that allows the bases to thepresented in the correct spatial relationship such that hybridization issubstantially similar to what is seen with a ribophosphate backbone,e.g., non-charged mimics of the ribophosphate backbone.

Antisense, siRNA, and other oligonucleotides useful in this inventioninclude, but are not limited to, oligonucleotides containing modifiedbackbones or non-natural internucleoside linkages. Oligonucleotideshaving modified backbones include those that retain a phosphorus atom inthe backbone and those that do not have a phosphorus atom in thebackbone. Modified oligonucleotides that do not have a phosphorus atomin their internucleoside backbone can also be considered to beoligonucleosides. Modified oligonucleotide backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotri-esters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, phosphoroselenate, methylphosphonate, orO-alkyl phosphotriester linkages, and boranophosphates having normal3′-5′ linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acidforms are also included. Representative United States patents that teachthe preparation of the above linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050.

In certain embodiments, modified oligonucleotide backbones that do notinclude a phosphorus atom therein have backbones that are formed byshort chain alkyl or cycloalkyl internucleoside linkages, mixedheteroatom and alkyl or cycloalkyl internucleoside linkages, or one ormore short chain heteroatomic or heterocyclic internucleoside linkages.These include, e.g., those having morpholino linkages (formed in partfrom the sugar portion of a nucleoside); siloxane backbones; sulfide,sulfoxide and sulfone backbones; formacetyl and thioformacetylbackbones; methylene formacetyl and thioformacetyl backbones; alkenecontaining backbones; sulfamate backbones; methyleneimino andmethylenehydrazino backbones; sulfonate and sulfonamide backbones; amidebackbones; and others having mixed N, O, S and CH₂ component parts.Representative United States patents that describe the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439.

The phosphorothioate backbone modification, where a non-bridging oxygenin the phosphodiester bond is replaced by sulfur, is one of the earliestand most common means deployed to stabilize nucleic acid drugs againstnuclease degradation. In general, it appears that PS modifications canbe made extensively to both siRNA strands without much impact onactivity (Kurreck, Eur. J. Biochem. 270:1628-44, 2003). In particularembodiments, the PS modification is usually restricted to one or twobases at the 3′ and 5′ ends. The boranophosphate linker can be used toenhance siRNA activity while having low toxicity (Hall et al., NucleicAcids Res. 32:5991-6000, 2004).

Other useful nucleic acids derivatives include those nucleic acidsmolecules in which the bridging oxygen atoms (those forming thephosphoester linkages) have been replaced with —S—, —NH—, —CH₂—, and thelike. In certain embodiments, the alterations to the antisense, siRNA,or other nucleic acids used will not completely affect the negativecharges associated with the nucleic acids. Thus, the present inventioncontemplates the use of antisense, siRNA, and other nucleic acids inwhich a portion of the linkages are replaced with, for example, theneutral methyl phosphonate or phosphoramidate linkages. When neutrallinkages are used, in certain embodiments, less than 80% of the nucleicacid linkages are so substituted, or less than 50% of the linkages areso substituted.

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C or m5c), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.Certain nucleobases are particularly useful for increasing the bindingaffinity of the oligomeric compounds of the invention, including5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi etal., eds., Antisense Research and Applications 1993, CRC Press, BocaRaton, pages 276-278). These may be combined, in particular embodiments,with 2′-O-methoxyethyl sugar modifications. United States patents thatteach the preparation of certain of these modified nucleobases as wellas other modified nucleobases include, but are not limited to, the abovenoted U.S. Pat. Nos. 3,687,808; 4,845,205; 5,130,302; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091;5,614,617; and 5,681,941.

Exemplary sugar modifications include modifications to the 2′-OH of theRNA sugar ring, which provides a convenient chemically reactive site.Exemplary modifications include the 2′-F and 2′-OMe modification, whichcan be restricted to less than 4 nucleotides per strand (Holen et al.,Nucleic Acids Res 31:2401-2407, 2003). The 2′-O-MOE is most effective insiRNA when modified bases are restricted to the middle region of themolecule (Prakash et al., J. Med. Chem. 48:4247-4253, 2005).

Modified oligonucleotides may also contain one or more substituted sugarmoieties. For example, the invention includes oligonucleotides thatcomprise one of the following at the 21 position: OH; F; O-, S-, orN-alkyl, O-alkyl-O-alkyl, O-, S-, or N-alkenyl, or O-, S- or N-alkynyl,wherein the alkyl, alkenyl and alkynyl may be substituted orunsubstituted C₁₋₁₀ alkyl or C₂₋₁₀ alkenyl and alkynyl. Particularlypreferred are O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃,O(CH₂)₂ON(CH₃)₂O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]═, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2′position: C₁₋₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl,O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃,SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. One modification includes2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl)or 2′-MOE) (Martin et al., Helv. Chim. Acta 78:486-504, 1995), i.e., analkoxyalkoxy group. Other modifications include2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE, and 2′-dimethylaminoethoxyethoxy (2′-DMAEOE). Additionalmodifications include 2′-methoxy (2′-O—CH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications may alsobe made at other positions on the oligonucleotide, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarsstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and5,700,920.

In other oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups, although the base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262. Further teaching of PNAcompounds can be found in Nielsen et al., Science 254, 1497-1500 (1991).

Particular embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular CH₂NHOCH₂, CH₂N(CH₃)OCH₂-(referred to as amethylene(methylimino) or MMI backbone), CH₂ON(CH₃)CH₂,CH₂N(CH₃)N(CH₃)CH₂, and ON(CH₃)CH₂CH₂ (wherein the native phosphodiesterbackbone is represented as OPOCH₂) of U.S. Pat. No. 5,489,677; the amidebackbones of U.S. Pat. No. 5,602,240; and the morpholino backbonestructures U.S. Pat. No. 5,034,506.

Treatment During Apheresis

The invention also features methods of treating a subject (e.g., asubject having or at risk of developing a renal disease) by contactingthe blood of the subject during extracorporeal apheresis methods withone or more of the agents of the invention (e.g., one or more agentsthat target one or more components of the JAK/STAT pathway, the TLPpathway, the NF-κB pathway, PKR pathway, the MAP kinase pathway, and/orthe TBK1/lKKe pathway).

Generally, apheresis includes the removal or withdrawal of blood fromthe subject's body, removal of one or more components from the blood,and transfusion of the remaining blood back into the subject's body. Inthe present invention, one or more agents of the invention (e.g., one ormore agents that target one or more components of the JAK/STAT pathway,the TLP pathway, the NF-κB pathway, PKR pathway, the MAP kinase pathway,and/or the TBK1/lKKe pathway) are contacted to the blood of a subjectduring apheresis.

Apheresis procedures and equipment are known in the art and can be usedin the present invention. In one example, once the patient's blood isremoved from a vein in the arm, the plasma is separated from the rest ofthe blood using a membrane plasma filter. Either the plasma or the bloodcan be contacted with one or more of the compositions of the inventionand then recombined and returned to the patient.

In yet another example, blood from the patient is circulatedextra-corporeally using standard apheresis equipment. The blood isseparated into the cellular elements (red blood cells, white blood cellsand platelets) and fluid (plasma) elements using differentialcentrifugation or a membrane filter. The plasma is then pumped throughthe targeted apheresis device where it can be contacted with one or moreof the compositions of the invention. Alternatively, the other bloodcomponents can be contacted with one or more of the compositions of theinvention. After the contacting step, the plasma is then mixed with thecellular blood elements and returned to the patient. In one embodiment,the pH of the blood is restored to normal biological levels prior toreturning to the subject.

Additional Therapies

The agents of the invention (e.g., one or more agents that target one ormore components of the JAK/STAT pathway, the TLP pathway, the NF-κBpathway, PKR pathway, the MAP kinase pathway, and/or the TBK1/lKKepathway) may be administered alone or in combination with other knowntherapies for the treatment of renal disease. For example, a subjecttreated with an agent of the invention (e.g., one or more agents thattarget one or more components of the JAK/STAT pathway, the TLP pathway,the NF-κB pathway, PKR pathway, the MAP kinase pathway, and/or theTBK1/lKKe pathway) may also be treated with a blood pressure medication,a steroid, and/or an immunosuppressive agent. Examples of therapeuticsinclude blood pressure medications (e.g., a diuretic (e.g.,chlorthalidone, chlorothiazide, furosemide, hydrochlorothiazide,indapamide, metolazone, amiloride hydrochloride, spironolactone,triamterene, bumetanide, or a combination thereof), an alpha adrenergicantagonist (e.g., alfuzosin, doxazosin, prazosin, terazosin, ortamsulosin, or a combination thereof), a central adrenergic inhibitor(e.g., clonidine, guanfacine, or methyldopa, or a combination thereof),an angiotensin converting enzyme (ACE) inhibitor (e.g., benazepril,captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril,quinapril, ramipril, or trandolapril, or combinations thereof), anangiotensin II receptor blocker (e.g., candesartan, eprosartan,irbesartan, losartan, olmesartan, telmisartan, or valsartan, orcombinations thereof), an alpha blocker (e.g., doxazosin, prazosin, orterazosin, or a combination thereof), a beta blocker (e.g., acebutolol,atenolol, betaxolol, bisoprolol, carteolol, metoprolol, nadolol,nebivolol, penbutolol, pindolol, propranolol, solotol, or timolol, or acombination thereof), a calcium channel blocker (e.g., amlodipine,bepridil, diltiazem, felodipine, isradipine, nicardipine, nifedipine,nisoldipine, or verapamil, or combination thereof), a vasodilator (e.g.,hydralazine or minoxidil, or combination thereof), and a renin inhibitor(e.g., aliskiren), or combinations thereof), a steroid (e.g., acorticosteroid, such as cortisone, prednisone, methylprednisolone, orprednisolone), or an anabolic steroid (anatrofin, anaxvar, annadrol,bolasterone, decadiabolin, decadurabolin, dehydropiandrosterone (DHEA),delatestryl, dianiabol, dihydrolone, durabolin, dymethazine,enoltestovis, equipose, gamma hydroxybutyrate, maxibolin, methatriol,methyltestosterone, parabolin, primobolin, quinolone, therabolin,trophobolene, and winstrol), or an immunosuppressive agent, such as aglucocorticoid, a cytostatic, an antibody, or an anti-immunophilinand/or mychophenolate mofetil (MMF), FK-506, azathioprine,cyclophosphamide, methotrexate, dactinomycin, antithymocyte globulin(ATGAM), an anti-CD20-antibody, a muromonoab-CD3 antibody, basilizimab,daclizumab, cyclosporin, tacrolimus, voclosporin, sirolimus, aninterferon, infliximab, etanercept, adalimumab, fingolimod, and/ormyriocin).

Administration and Dosage

Agents of the invention (e.g., one or more agents that target one ormore components of the JAK/STAT pathway, the TLP pathway, the NF-κBpathway, PKR pathway, the MAP kinase pathway, and/or the TBK1/lKKepathway) may be administered in methods of the invention in atherapeutically effective amount. In some examples, a therapeuticallyeffective amount of an agent of the invention (e.g., one or more agentsthat target one or more components of the JAK/STAT pathway, the TLPpathway, the NF-κB pathway, PKR pathway, the MAP kinase pathway, and/orthe TBK1/lKKe pathway) includes an amount, per dose, in the range ofabout 0.01 mg/kg to about 1000 mg/kg (such as about 0.01 mg/kg to 1000mg/kg, 0.05 mg/kg to 500 mg/kg, 0.1 mg/kg to 1000 mg/kg, about 10 mg/kgto 500 mg/kg, about 10 mg/kg to 100 mg/kg, about 50 mg/kg to 500 mg/kg,or about 100 mg/kg to 1000 mg/kg). Administration can be accomplished bysingle or multiple doses and depends on the specific agent beingadministered and the administration route. Doses are determined for eachparticular case using standard methods in accordance with factors uniqueto the patient, including age, weight, general state of health, andother factors that can influence the efficacy of the compound(s) of theinvention. The dose required will vary from subject to subject dependingon the species, age, weight and general condition of the subject, theparticular therapeutic agent being used and its mode of administration.For example, preferred doses can be 0.1-1 mg/kg by inhalation, desirably0.5-10 mg/kg per day by oral administration, and desirably 0.1-1 mg/kgbody weight per day by intravenous administration. Generally, dosagelevels of an agent of the invention of between 0.1 μg/kg to 100 mg/kg ofbody weight are administered daily, weekly, monthly, or yearly as asingle dose or divided into multiple doses (e.g., 2-12 doses per day,week, month, or year), or as needed. Preferably, the general dosagerange is between 250 μg/kg to 50.0 mg/kg of body weight per day. Widevariations in the needed dosage are to be expected in view of thediffering efficiencies of the various routes of administration. Forinstance, oral administration generally would be expected to requirehigher dosage levels than administration by intravenous injection.Variations in these dosage levels can be adjusted using standardempirical routines for optimization, which are well known in the art. Ingeneral, the precise therapeutically effective dosage will be determinedby the attending physician in consideration of the above-identifiedfactors. It will be appreciated that these dosages are examples only,and an appropriate dose can be determined by one of ordinary skill inthe art using only routine experimentation.

Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acid salts includeacetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,bisulfate, butyrate, citrate, camphorate, camphorsulfonate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptanoate, glycerophosphate, glycolate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate,palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, salicylate, succinate, sulfate, tartrate,thiocyanate, tosylate and undecanoate. Other acids, such as oxalic,while not in themselves pharmaceutically acceptable, may be employed inthe preparation of salts useful as intermediates in obtaining thecompounds of the invention and their pharmaceutically acceptable acidaddition salts.

Pharmaceutical formulations of a therapeutically effective amount of anagent of the invention (e.g., one or more agents that target one or morecomponents of the JAK/STAT pathway, the TLP pathway, the NF-κB pathway,PKR pathway, the MAP kinase pathway, and/or the TBK1/lKKe pathway), orpharmaceutically acceptable salts thereof, can be administered orally,parenterally (e.g., as an intramuscular, intraperitoneal, intravenous,intraarterial, or subcutaneous injection), by inhalation, intradermally,by optical drops, by implant, nasally, vaginally, rectally,sublingually, or topically, and may be in admixture with apharmaceutically acceptable carrier adapted for the route ofadministration. The choice of vehicle and the content of activesubstance in the vehicle are generally determined in accordance with thesolubility and chemical properties of the product, the particular modeof administration, and the provisions to be observed in pharmaceuticalpractice. For example, excipients such as lactose, sodium citrate,calcium carbonate, and dicalcium phosphate and disintegrating agentssuch as starch, alginic acids, and certain complex silicates combinedwith lubricants (e.g., magnesium stearate, sodium lauryl sulfate, andtalc) may be used for preparing tablets. To prepare a capsule, it isadvantageous to use lactose and high molecular weight polyethyleneglycols. When aqueous suspensions are used, they may contain emulsifyingagents that facilitate suspension. Diluents such as sucrose, ethanol,polyethylene glycol, propylene glycol, glycerol, chloroform, or mixturesthereof may also be used.

An agent of the invention (e.g., an agent that targets one or morecomponents of the JAK/STAT pathway, the TLP pathway, the NF-κB pathway,PKR pathway, the MAP kinase pathway, and/or the TBK1/lKKe pathway) mayalso be administered in the form of liposome delivery systems, such assmall unilamellar vesicles, large unilamellar vesicles, andmultilamellar vesicles. Liposomes can be formed from a variety oflipids, such as cholesterol, stearylamine, or phosphatidylcholines.

Methods well known in the art for making formulations are found, forexample, in Remington's Pharmaceutical Sciences (18^(th) edition), ed.A. Gennaro, 1990, Mack Publishing Company, Easton, Pa. Compositionsintended for oral use may be prepared in solid or liquid forms accordingto any method known to the art for the manufacture of pharmaceuticalcompositions. The compositions may optionally contain sweetening,flavoring, coloring, perfuming, and/or preserving agents in order toprovide a more palatable preparation. Solid dosage forms for oraladministration include capsules, tablets, pills, powders, and granules.In such solid forms, the active agent is admixed with at least one inertpharmaceutically acceptable carrier or excipient. These may include, forexample, inert diluents, such as calcium carbonate, sodium carbonate,lactose, sucrose, starch, calcium phosphate, sodium phosphate, orkaolin. Binding agents, buffering agents, and/or lubricating agents(e.g., magnesium stearate) may also be used. Tablets and pills canadditionally be prepared with enteric coatings.

The pharmaceutically acceptable compositions of this invention may beorally administered in any orally acceptable dosage form including, butnot limited to, capsules, tablets, aqueous suspensions or solutions. Inthe case of tablets for oral use, carriers commonly used include lactoseand corn starch. Lubricating agents, such as magnesium stearate, arealso typically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added. Liquid dosage forms fororal administration include pharmaceutically acceptable emulsions,solutions, suspensions, syrups, and soft gelatin capsules. These formscontain inert diluents commonly used in the art, such as water or an oilmedium. Besides such inert diluents, compositions can also includeadjuvants, such as wetting agents, emulsifying agents, and suspendingagents.

Formulations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, or emulsions. Examples of suitablevehicles include propylene glycol, polyethylene glycol, vegetable oils,gelatin, hydrogenated naphthalenes, and injectable organic esters, suchas ethyl oleate. Such formulations may also contain adjuvants, such aspreserving, wetting, emulsifying, and dispersing agents. Biocompatible,biodegradable lactide polymer, lactide/glycolide copolymer, orpolyoxyethylene-polyoxypropylene copolymers may be used to control therelease of the compounds. Other potentially useful parenteral deliverysystems for the agents of the invention include ethylene-vinyl acetatecopolymer particles, osmotic pumps, implantable infusion systems, andliposomes.

Liquid formulations can be sterilized by, for example, filtrationthrough a bacteria-retaining filter, by incorporating sterilizing agentsinto the compositions, or by irradiating or heating the compositions.Alternatively, they can also be manufactured in the form of sterile,solid compositions, which can be dissolved in sterile water or someother sterile injectable medium immediately before use.

Compositions for rectal or vaginal administration are preferablysuppositories, which may contain, in addition to active substances,excipients such as cocoa butter or a suppository wax. Compositions fornasal or sublingual administration are also prepared with standardexcipients known in the art. Formulations for inhalation may containexcipients, for example, lactose, or may be aqueous solutionscontaining, for example, polyoxyethylene-9-lauryl ether, glycocholateand deoxycholate, or may be oily solutions for administration in theform of nasal drops or spray, or as a gel.

The amount of active agent of the invention can be varied. One skilledin the art will appreciate that the exact individual dosages may beadjusted somewhat depending upon a variety of factors, including theingredient being administered, the time of administration, the route ofadministration, the nature of the formulation, the rate of excretion,the nature of the subject's conditions, and the age, weight, health, andgender of the patient. In addition, the severity of the conditiontargeted by an agent of the invention will also have an impact on thedosage level.

An agent of the invention can be administered in a sustained releasecomposition, such as those described in, for example, U.S. Pat. Nos.5,672,659 and 5,595,760, hereby incorporated by reference, or as aliposomal formulation. The use of immediate or sustained releasecompositions depends on the type of condition being treated. If thecondition consists of an acute or over-acute disorder, a treatment withan immediate release form will be preferred over a prolonged releasecomposition. Alternatively, for preventative or long-term treatments, asustained released composition will generally be preferred.

Where sustained release administration of the agent (e.g., a compound,an antibody, peptide, or nucleic acid molecule) is desired in aformulation with release characteristics suitable for the treatment ofany disease or disorder requiring administration of the agent (e.g.,treatment of renal disease), microencapsulation of the agent iscontemplated. Microencapsulation of recombinant proteins for sustainedrelease has been successfully performed with human growth hormone(rhGH), interferon-(rhIFN-), interleukin-2, and MN rgp120 (Johnson etal., Nat. Med. 2: 795-799, 1996; Yasuda, Biomed. Ther. 27: 1221-1223,1993; Hora et al., Bio/Technology 8: 755-758 1990; Cleland, “Design andProduction of Single Immunization Vaccines Using PolylactidePolyglycolide Microsphere Systems,” in “Vaccine Design: The Subunit andAdjuvant Approach,” Powell and Newman, Eds., Plenum Press: New York, pp.439-462, 1995; WO 97/03692; WO 96/40072; WO 96/07399; and U.S. Pat. No.5,654,010, hereby incorporated by reference).

The sustained-release formulations may also include those developedusing poly-lactic-coglycolic acid (PLGA) polymer. The degradationproducts of PLGA, lactic and glycolic acids, can be cleared quickly fromthe human body. Moreover, the degradability of this polymer can beadjusted from months to years depending on its molecular weight andcomposition (see, e.g., Lewis, “Controlled release of bioactive agentsfrom lactide/glycolide polymer,” in M. Chasin and Dr. Langer (Eds.),Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: NewYork, pp. 1-41, 1990)).

An agent of the invention can be prepared in any suitable manner. Theagent may be isolated from naturally-occurring sources, or producedsynthetically, identified from a library, or produced by a combinationof these methods.

Assessment of Therapy

After therapeutic treatment with the compositions of the inventiondescribed herein, the efficacy of the treatment may be assessed by anumber of methods, such as assays that measure hypoalbuminemia (lowserum albumin) in the blood, a reduction in symptoms of hyperlipidemiaand hypertension (high blood pressure), a reduction in edema (fluidretention), a reduction in inflammation in the kidney (e.g., in thenephron), a reduction in protein in the urine (proteinuria), a reductionof blood in the uring (hematuria), and/or an increase in renal function.Tests that can be performed include urinalysis, blood tests (for, e.g.,cholesterol), and kidney biopsy (e.g., a reduction in sclerosis(scarring) of the glomerulus). Efficacy may also be indicated by animprovement in or resolution of one or more symptoms of renal disease orin a reduced need of, or frequency of, dialysis.

Diagnostics

A patient that may be in need of one or more of the treatment methodsdescribed herein can be identified by detecting one or more APOL1 riskalleles in the patient (e.g., one or more of the G1a, G1b, G1, G2, or G3risk alleles described herein). Preferably, the patient includes two ormore APOL1 risk alleles. In other embodiments, the subject is of Africanor Hispanic ancestry. For example, subjects of African or Hispanicancestry that have at least two (or more) of APOL1 gene risk allelesexhibit a significantly increased risk of developing renal disease.Thus, these subjects may be assayed for the presence of a wild typeallele (relative to an APOL1 gene risk allele) as a means fordetermining whether the subject has a moderate or increased risk ofrenal disease. For example, a subject that is heterozygous at a givenlocus for one or more of the APOL1 gene risk alleles may have a greaterrisk of renal disease relative to a subject lacking any APOL1 gene riskalleles, and thus may be more likely to benefit from treatment using oneor more of the methods or compositions described herein. A subject thatis homozygous at a given locus for one or more APOL1 gene risk allelesmay have a greater risk of renal disease, relative to a subject that isheterozygous for an APOL1 gene risk allele at that locus or a subjectthat lacks any risk alleles in an APOL1 gene, and thus may be morelikely to benefit from treatment using one or more of the methods orcompositions described herein. The presence of two or more (e.g., three,four, or more) risk alleles at different loci further increases thelikelihood of renal disease in a subject. Thus a subject having two ormore (e.g., three, four, or more) risk alleles at different loci islikely to benefit from treatment using one or more of the methods orcompositions described herein.

Thus, the present invention also features a method of diagnosing asubject that is likely to benefit from one or more of the treatmentmethods described herein by using any number of methods known in theart. For example, these can include, genomic sequencing assay,polymerase chain reaction assay, fluorescence in situ hybridizationassay, immunoassay, or using one or more of the methods or kitsdescribed in, e.g., PCT/US2011/032924, which is incorporated herein byreference, to identify the presence or absence of one or more APOL1 riskalleles in the subject. Once the subject has been identified as havingone or more (or two, three, or four, or more) APOL1 risk alleles at oneor more loci, and thus to have or to be at a greater risk of developinga renal disease, the subject may be administered one or more of thecompositions of the invention in order to treat (prophylactically ortherapeutically) the renal disease or to reduce one or more of thesymptoms of the renal disease.

The invention also provides for a diagnostic test kit for detecting thepresence of one or more APOL1 risk alleles. The invention also providesa kit for treating a subject once a diagnosis that the subject has or isat risk of developing renal disease has been made. The kit may includethe diagnostic reagents and instructions for detecting the presence ofan APOL1 risk allele only or the diagnostic reagents and one or moreagents of the invention (e.g., one or more agents that target one ormore components of the JAK/STAT pathway, the TLP pathway, the NF-κBpathway, PKR pathway, the MAP kinase pathway, and/or the TBK1/lKKepathway) for treating the renal disease and, optionally, instructionsfor performing the diagnosis and the treatment methods, or only agentsof the invention for treating the renal disease and, optionally,instructions for the treatment methods. The kit may also include one ormore other therapeutic agents, such as a blood pressure medication, asteroid, or an antiinflammatory agent.

EXAMPLES

In order to make the methods of the invention clearer, the followingexamples are presented. These examples are only for illustrativepurposes and should not be interpreted in any way as limitations on thecompositions and uses of this invention.

Example 1: Severe and Widespread Liver Injury Caused by G1 Risk Variant

Wild type and risk variant APOL1 cDNA were injected into mice viahydrodynamic gene delivery (FIG. 1). The cDNA was taken up and expressedby the liver. Mice injected with wild type APOL1 showed only subtlesigns of liver injury, whereas mice injected with a risk variant APOL1had severe and widespread necrosis, indicating greater toxicity of therisk variants. These findings support an unusual gain-of-function modeldespite recessive mode of inheritance, a situation rare in biology andto our knowledge unique for a common disease.

Example 2: Variation in Human APOL1 Expression Between Individuals

Not all individuals with the APOL1 risk genotype develop kidney disease.If APOL1 mediated renal disease is caused by a gain-of-functionmutation, then expression differences may determine which individualswill develop kidney disease. Gene expression profiling studies oflymphoblasts from Africans and Europeans (FIG. 2) shows that Africanshave on average higher APOL1 gene expression levels than Europeans. Morestriking are the expression differences between individuals within eachgroup.

Example 3: Inflammatory Factors Cause Log-Fold Upregulation of APOL1

Interferons cause log-fold upregulation of APOL1 gene expression inseveral cell types. Both type 1 (alpha and beta) interferons and type 2(gamma) interferon greatly amplifies APOL1 expression in bothendothelial cells and podocytes. FIG. 3A shows interferon gammaupregulates APOL1 in endothelial cells by up to 200-fold, whereasinterferon beta (FIG. 3B) upregulates APOL1 in endothelial cells by upto 30-fold.

Example 4: JAK Inhibitors Block the Potentially Pathologic Upregulationof APOL1

JAK inhibitors are in clinical testing for autoimmune disease,transplant immunosuppression, and myelodysplastic syndromes. They areselective, rather than specific, for individual JAK inhibitors. Forexample, WHI-P131 targets JAK3, INCB018424 (ruxolitinib) targets JAK1and JAK2, and TG101348 targets JAK2. APOL1 mRNA expression was measuredin endothelial cells treated with interferon beta and various JAKinhibitors. As shown in FIGS. 4A-4C, the JAK inhibitors block thepotentially pathologic upregulation of APOL1 caused by interferon betatreatment. Similar effects were also seen in podocytes.

Example 5: TLR, Rig, and Intracellular DNA Sensors Also Upregulate APOL1

HIV is a powerful inducer of APOL1 renal disease in individuals with therenal risk genotype. Other viruses such as parvovirus have beenassociated with FSGS. Viral sensing stimulates APOL1 expression. Cellshave extracellular, endosomal, and cytoplasmic viral sensors such asTLRs, Rig-L1, and others. These genes are potential targets for reducingAPOL1 expression. FIGS. 5A-5B show large increases in APOL1 expressionin endothelial cells (FIG. 5A) and podocytes (FIG. 5B) after treatmentwith TLR agonists. FIGS. 6A-6B show increases in APOL1 expression inendothelial cells (FIG. 6A) and podocytes 9 FIG. 6B) treated with short,5′ PPP dsRNA, an agonist of cytoplasmic RIG-L1 receptors. FIG. 7 showsan increase in APOL1 mRNA expression in endothelial cells treated withLPS-free bacterial DNA, with or without the transfection agent Lyovec.

Example 6: JAK Inhibitors, Bx795 (a TBK1/lKKe Inhibitor), andChloroquine Block the TLR3-Stimulated APOL1 Upregulation

Endothelial cells were treated with the TLR3 agonist poly(I:C) and withJAK inhibitors (FIGS. 8A-8D), Bx795 (a TBK1/lKKe inhibitor) (FIG. 9),and chloroquine (FIG. 10). The JAK inhibitors reduced the poly(I:C)induced increased in APOL1 expression, as did Bx795, and chloroquine.These results demonstrate that blocking diverse pathways may be astrategy to reduce pathogenic APOL1 expression and provides not onlytreatment options in patients with an APOL1 risk allele, but also amultitude of targets for new drug development. The TBK1/lKKe complex isused by both TLR and Rig-like receptor signaling mechanisms, andtherefore offers a broader target for inhibition of APOL1 expressionthan either pathway alone. Chloroquine and similar class of compoundsmay be particularly useful for inhibiting APOL1 expression when thatexpression is driven by endosomal pattern recognition receptors such asTLR3, TLR7, TLR8, TLR9, and in some cases TLR4. Even though TLR3expression is described above as an example, these strategies are likelyto be effective in blocking APOL1 expression driven by multipleinflammatory stimuli.

Example 7: NF-κB Inhibitors, PKR Inhibitors, and MAP Kinase Inhibitorsall Block Increased APOL1 Expression Driven by Inflammatory StimuliInduced by a TLR3 Agonist

Endothelial cells were treated with the TLR3 agonist poly(I:C) and withNF-κB inhibitors (FIG. 11), PKR inhibitors (FIG. 12), and MAP kinaseinhibitors (FIG. 13).

APOL1 expression stimulated by poly(I:C) could be inhibited by 2different NF-kB inhibitors. The first inhibitor, Bay 11-7085, blocksphosphorylation of IkBa, thereby preventing translocation of NF-kBsubunits to the nucleus where they regulate gene expression (in thiscase APOL1). The second, 6-amino-4-(4-phenoxyphenylethylamino)quinazoline (abbreviated 6A4Q), inhibits NF-kB transcriptionalactivation of target genes. Inhibition at two different steps of theNF-kB activation of APOL1 expression suggests that any of severalhundred other known inhibitors of NF-kB signaling may be useful insuppressing blocked poly(I:C) induced APOL1 upregulation.

To block the PKR pathway, an imidazolo-oxindole PKR inhibitor (chemicalformula C₁₃H₈N₄OS-EMD chemicals) was used. This inhibitor has thestructure

and completely blocked poly(I:C) induced APOL1 upregulation.

The MAP kinase (p38) inhibitor SB203580 also inhibited blocked poly(I:C)induced APOL1 upregulation in endothelial cells, consistent withexisting chromatin immunoprecipitation data showing AP-1 binding sitesin the APOL1 promoter. In podocytes, the JNK inhibitor SP600125inhibited APOL1 expression by 50% (not shown).

Example 8: shRNA Knockdown Experiments

To validate pharmacologic targets that may regulate pathogenic APOL1expression, we used shRNA to determine specific genes that mediate theAPOL1 upregulation induced by poly(I:C). These experiments allowed us toconfirm targets identified above by pharmacologic methods and toidentify additional known targets in these same pathways. Reduced APOL1expression (which was induced by poly(I:C) stimulus) was seen withknockdown of JAKs, STATs, IRFs, NF-κB pathway genes (NEMO, REL-A), andgenes involved in TLR signaling (TLR3, Ticam, TBK1). The data shown inFIG. 14 validates several pathways identified by the pharmacologicalexperiments described above and identifies additional pathways that canbe targeted to decrease pathogenic APOL1 expression.

OTHER EMBODIMENTS

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described herein. The term “comprises” means “includes.”In case of conflict, the present specification, including explanationsof terms, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure that come within known or customary practice withinthe art to which the invention pertains and may be applied to theessential features hereinbefore set forth.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindependent publication or patent application was specifically andindividually indicated to be incorporated by reference in theirentirety.

In view of the many possible embodiments to which the principles of thedisclosure may be applied, it should be recognized that the illustratedembodiments are only examples and should not be taken as limiting thescope of the invention. Rather, the scope of the invention is defined bythe following claims. We therefore claim as our invention all that comeswithin the scope and spirit of these claims.

The invention claimed is:
 1. A method of treating or reducing thelikelihood of developing focal segmental glomerulosclerosis (FSGS) in asubject who has not had a kidney transplant, said subject having beenidentified as being at risk of developing the renal disease based on thepresence of: (i) two copies of the G1 allele of APOL1; (ii) two copiesof the G2 allele of APOL1; or (iii) one copy of the G1 allele of APOL1and one copy of the G2 allele of APOL1, wherein the method comprisesadministering to the subject an antagonist of Janus kinase 1 (JAK1),Janus kinase 2 (JAK2), or Janus kinase 3 (JAK3).
 2. The method of claim1, wherein said antagonist is selected from the group consisting ofINCB-018424, TG101348, WHI-P131, and CP-690550.
 3. The method of claim1, wherein said antagonist inhibits JAK1.
 4. The method of claim 1,wherein FSGS specifically leads to damage of the kidneys.
 5. The methodof claim 1, further comprising, prior to administering said antagonist,testing said subject for the presence of the APOL1 allele.
 6. The methodof claim 1, wherein said subject has one copy of the G1 allele of APOL1and one copy of the G2 allele of APOL1.
 7. The method of claim 1,wherein said subject is of African or Hispanic ancestry.
 8. The methodof claim 7, wherein said subject is an African-American subject.
 9. Themethod of claim 1, further comprising administering to said subject atherapeutic agent.
 10. The method of claim 9, wherein said therapeuticagent is a blood pressure medication, a steroid, or an immunosuppressiveagent.
 11. The method of claim 10, wherein: i) said blood pressuremedication is a diuretic, wherein preferably said diuretic is selectedfrom chlorthalidone, chlorothiazide, furosemide, hydrochlorothiazide,indapamide, metolazone, amiloride hydrochloride, spironolactone,triamterene, bumetanide, and combinations thereof; an alpha adrenergicantagonist, wherein preferably said alpha adrenergic antagonist isselected from alfuzosin, doxazosin, prazosin, terazosin, or tamsulosin,and combinations thereof; a central adrenergic inhibitor, whereinpreferably said central adrenergic inhibitor is selected from clonidine,guanfacine, methyldopa, and combinations thereof; an angiotensinconverting enzyme (ACE) inhibitor, wherein said ACE inhibitor isselected from benazepril, captopril, enalapril, fosinopril, lisinopril,moexipril, perindopril, quinapril, ramipril, trandolapril, andcombinations thereof; an angiotensin II receptor blocker, wherein saidangiotensin II receptor blocker is selected from candesartan,eprosartan, irbesartan, losartan, olmesartan, telmisartan, valsartan,and combinations thereof; an alpha blocker, wherein said an alphablocker is selected from doxazosin, prazosin, terazosin, andcombinations thereof; a beta blocker, wherein said beta blocker isselected from acebutolol, atenolol, betaxolol, bisoprolol, carteolol,metoprolol, nadolol, nebivolol, penbutolol, pindolol, propranolol,solotol, timolol, and combinations thereof; a calcium channel blocker,wherein said calcium channel blocker is selected from amlodipine,bepridil, diltiazem, felodipine, isradipine, nicardipine, nifedipine,nisoldipine, verapamil, and combinations thereof); a vasodilator,wherein said vasodilator is selected from hydralazine, minoxidil, andcombinations thereof; and a renin inhibitor, such as aliskiren, andcombinations thereof; and/or ii) said steroid is selected from acorticosteroid, such as cortisone, prednisone, methylprednisolone,prednisolone, and combinations thereof; an anabolic steroid, such asanatrofin, anaxvar, annadrol, bolasterone, decadiabolin, decadurabolin,dehydropiandrosterone (DHEA), delatestryl, dianiabol, dihydrolone,durabolin, dymethazine, enoltestovis, equipose, gamma hydroxybutyrate,maxibolin, methatriol, methyltestosterone, parabolin, primobolin,quinolone, therabolin, trophobolene, winstrol, and combinations thereof;and/or iii) said immunosuppressive agent is a glucocorticoid, acytostatic, an antibody, or an anti-immunophilin and/or mychophenolatemofetil (MMF), FK-506, azathioprine, cyclophosphamide, methotrexate,dactinomycin, antithymocyte globulin (ATGAM), an anti-CD20-antibody, amuromonoab-CD3 antibody, basilizimab, daclizumab, cyclosporin,tacrolimus, voclosporin, sirolimus, an interferon, infliximab,etanercept, adalimumab, fingolimod, myriocin, and combinations thereof.12. The method of claim 1, further comprising transplanting a donorkidney into the subject after administration of the antagonist, wherein,prior to the transplantation, the donor kidney is contacted with theantagonist.
 13. The method of claim 1, wherein the subject is in need ofa kidney transplant.
 14. The method of claim 1, wherein the methodfurther comprises, after administration of the antagonist, transplantinga donor kidney into the subject.
 15. The method of claim 1, wherein thesubject is a human.
 16. The method of claim 1, wherein said subject ishomozygous for the G1 allele of APOL1.
 17. The method of claim 1,wherein the subject is homozygous for the G2 allele of APOL1.
 18. Themethod of claim 1, wherein administration of said antagonist decreasesthe level of said polypeptide in said subject relative to the level ofsaid polypeptide in a reference subject having one or two said APOL1alleles that is not administered said antagonist.
 19. The method ofclaim 1, wherein said antagonist inhibits JAK2.
 20. The method of claim1, wherein said antagonist inhibits JAK3.
 21. The method of claim 5,wherein said testing comprises a genomic sequencing assay, polymerasechain reaction assay, fluorescence in situ hybridization assay, or animmunoassay.